Multiplexed immunohistochemistry using recombinant antibodies with epitope tags

ABSTRACT

The present disclosure is directed to epitope-tagged antibodies, as well as methods of employing the epitope-tagged antibodies for detecting one or more targets in a biological sample, e.g. a tissue sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of PCT application PCT/US2017/021157, filed Mar.7, 2017, which claims the benefit of U.S. Provisional Patent Application62/461,651 filed Feb. 21, 2017, and the benefit of U.S. ProvisionalPatent Application 62/418,667 filed Nov. 7, 2016, and the benefit U.S.Provisional Patent Application 62/305,440 filed Mar. 8, 2016, thedisclosures of which are hereby incorporated by reference herein intheir entireties.

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Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing is the form of an ASCII-complianttext file (entitled “FILENAME,” created on DATE, and FILESIZE bytes insize) is submitted concurrently with the instant application, and theentire contents of the Sequence Listing are incorporated herein byreference.

BACKGROUND OF THE DISCLOSURE A. Field of the Subject Disclosure

The present disclosure provides for antibodies comprising epitope tags.

B. Description of the Related Art

Cell staining methods, including immunohistochemistry (IHC) and in situhybridization analysis (ISH), are useful tools in histological diagnosisand the study of tissue morphology. IHC employs specific binding agentsor moieties, such as antibodies, to detect an antigen of interest thatmay be present in a tissue sample. IHC is widely used in clinical anddiagnostic applications, such as to diagnose particular disease statesor conditions. For example, particular cancer types can be diagnosedbased on the presence of a particular marker molecule in a sampleobtained from a subject. IHC is also widely used in basic research tounderstand biomarker distribution and localization in different tissues.Biological samples also can be examined using in situ hybridizationtechniques, such as silver in situ hybridization (SISH), chromogenic insitu hybridization (CISH) and fluorescence in situ hybridization (FISH),collectively referred to as ISH. ISH is distinct from IHC in that ISHdetects nucleic acids in tissue whereas IHC detects proteins in tissue.

Characterization and quantitation of the multitude of proteins expressedby an organism's genome are the focus of proteomics. Multipleximmunohistochemistry (MIHC) represents a major unmet technological needto detect and analyze multivariate protein targets in paraffin-embeddedformalin-fixed tissues with broad applications in research anddiagnostics. Multiplex immunohistochemistry (MIHC) techniques areattempting to address the need for detecting and analyzing multivariateprotein targets in formalin-fixed, paraffin-embedded tissues. EffectiveMIHC techniques have broad applications in research and diagnostics.However, there are few, if any, efficient and reproducible methods thatallow simultaneous and quantitative detection of multiple proteintargets in tissues.

Epitope tagging is a recombinant DNA method for making a gene productimmunoreactive to an already existing antibody (Jarvik and Telmer, Annu.Rev. Genet. 32:601-618, 1998). Typically, the process involves insertinga nucleotide sequence encoding a peptide tag into a gene of interest andexpressing the gene in an appropriate host. The protein can then bedetected and/or purified by virtue of its interaction with the antibodyspecific to the epitope tag. This approach can elucidate the size of thetagged protein as well as its abundance, cellular location,posttranslational modifications and interactions with other proteins. Inparticular, antibodies recognizing the peptide tag facilitatepurification and/or isolation of tagged proteins.

BRIEF SUMMARY OF THE DISCLOSURE

In one aspect of the present disclosure is a recombinant antibody thatexpresses an epitope tag (referred to herein as an “epitope-taggedantibody”). In another aspect of the present disclosure is an antibodycomprising at least one epitope tag construct. In some embodiments, theepitope tag construct comprises tandem epitope tag repeats separated byspacers. In some embodiments, the epitope tag construct has the generalstructure -[Spacer]-[Epitope Tag], which may be repeated one or moretimes (e.g. from 1 to 12 times). In some embodiments, the at least oneepitope tag construct is expressed at a C-terminal end of a heavy chainconstant region or at a C-terminal end of a light chain constant regionof an antibody. In other embodiments, the at least one epitope tagconstruct is expressed at both the C-terminal end of the heavy chainconstant region and at the C-terminal end of the light chain constantregion.

In some embodiments, the epitope tag is selected from the groupconsisting of V5, HA, VSV, AU1, AU5, OLLAS, E, E2, KT3, AU1 and OLLAS.In some embodiments, the epitope tag comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, and SEQ ID NO: 9.

In some embodiments, the epitope tag construct comprises between 2 and 8epitope tags. In some embodiments, the epitope tag construct comprises 4epitope tags. In some embodiments, the epitope tag construct comprises 5epitope tags. In some embodiments, the epitope-tagged antibody comprisesbetween 4 and 10 epitope tags. In some embodiments, a ratio of a numberof epitope tags incorporated at the C-terminal end of a heavy chainconstant region to a number of epitope tags incorporated at theC-terminal end of a light chain constant region ranges from about 2:1 toabout 1:2. In some embodiments, a number of epitope tags incorporated atthe C-terminal end of a heavy chain constant region ranges from between2 to 6 epitope tags, and a number of epitope tags incorporated at theC-terminal end of a light chain constant region ranges from between 0 to4 epitope tags.

In some embodiments, at least a portion of an amino acid sequenceconstituting at least one spacer of the epitope tag construct isselected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. In some embodiments, atleast a portion of an amino acid sequence constituting a first spacer ofthe epitope tag construct is selected from one of SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, and wherein atleast a portion of an amino acid sequence constituting a second spacerof the epitope tag construct is selected from another one of SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.

In some embodiments, a molecular weight of an epitope tag constructrange from between about 5 g/mol to about 35 g/mol. In some embodiments,a combined molecular weight of all epitope tag constructs of anyepitope-tagged antibody ranges from between about 5 g/mol to about 50g/mol. In some embodiments, a combined molecular weight of all epitopetag constructs of any epitope-tagged antibody is less than 30% of themolecular weight of the corresponding native antibody.

In some embodiments, the epitope-tagged antibody is specific to a targetselected from the group consisting of CD3, CD8, CD20, CD68, PDL1, FoxP3,HER2, and EGFR2. In some embodiments, the epitope tag constructcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32. Insome embodiments, the antibody comprises two epitope tag constructsconjugated to terminal ends of a heavy chain constant region and a lightchain constant region, wherein each of the two epitope tag constructscomprise the same sequence, wherein the sequence is selected from thegroup consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, andSEQ ID NO: 32.

In another aspect of the present disclosure is a kit comprising anepitope-tagged antibody, such as those described herein, and detectionreagents for detecting the epitope-tagged antibody. In some embodiments,the detection reagents are anti-tag antibodies specific for an expressedepitope tag of the epitope-tagged antibody, and where the anti-tagantibody comprises a detectable moiety. In some embodiments, thedetectable moiety is a fluorophore. In some embodiments, the detectablemoiety is an enzyme, and additional chromogenic substrates for theenzyme are included within the detection kit. In some embodiments, thekit further comprises at least one unmodified antibody of antibodyconjugate, and further detection reagents to detect the at least oneunmodified antibody of antibody conjugate. In some embodiments, the kitfurther comprises at least one nucleic acid probe and yet furtherdetection reagents to detect the at least one nucleic acid probe.

In another aspect of the present disclosure is a method for detecting atarget in a sample (e.g. a tissue sample), comprising contact the samplewith an epitope-tagged antibody (such as those disclosed herein) anddetecting the target using the expressed epitope tags of the antibody.In some embodiments, the sample is contacted with two or more differentepitope-tagged antibodies, where each epitope-tagged antibody isspecific for a different target and wherein each antibody expressesdifferent epitope tags. In some embodiments, the sample is contactedsimultaneously with the two or more different epitope-tagged antibodies.In some embodiments, the method further comprises contacting the samplewith detection reagents for detecting the different epitope-taggedantibodies. In some embodiments, the detection reagents are anti-tagantibodies specific for the different expressed epitope tags of theepitope-tagged antibodies. In some embodiments, the anti-tag antibodiesspecific for the different expressed epitope tags of the epitope-taggedantibodies are introduced simultaneously. In some embodiments, each ofthe anti-tag antibodies are conjugated to fluorophores. In someembodiments, the method further comprises contacting the sample with oneor more unmodified antibodies or antibody conjugates. In someembodiments, the one or more unmodified antibodies or antibodyconjugates are introduced to the sample prior to the introduction of theepitope-tagged antibody.

In another aspect of the present disclosure is a method of multiplexdetection comprising (i) simultaneously contacting a tissue sample withtwo or more epitope-tagged antibodies, wherein each epitope-taggedantibody comprises different epitope tags; and (ii) simultaneouslycontacting the tissue sample with two or more anti-tag antibodies,wherein each anti-tag antibody is specific to one of the epitope-taggedantibodies, and where each anti-tag antibody comprises a differentdetectable moiety. In some embodiments, the two or more epitope-taggedantibodies applied as a mixture; and where the two or more anti-tagantibodies are applied as a mixture. In some embodiments, theepitope-tagged antibodies comprise an epitope tag selected from thegroup consisting of V5, HA, VSV, AU1, AU5, OLLAS, E, E2, KT3, AU1, andOLLAS. In some embodiments, the epitope-tagged antibodies comprise anepitope tag having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:9. In some embodiments, the epitope-tagged antibodies comprise between 4and 10 epitope tags.

In some embodiments, the anti-tag antibodies are conjugated to afluorescent moiety. In some embodiments, the fluorescent moiety isselected from the group consisting of fluorescent moiety selected fromxanthene derivatives, cyanine derivatives, squaraine derivatives,naphthalene derivatives, coumarin derivatives, oxadiazole derivatives,anthracene derivatives, pyrene derivatives, oxazine derivatives,acridine derivatives, arylmethine derivatives, and tetrapyrrolederivatives.

In some embodiments, the method further comprises contacting the tissuesample with at least one unmodified antibody. In some embodiments, thestep of contacting the tissue sample with at least one unmodifiedantibody occurs before the tissue sample is contacted with the two ormore epitope-tagged antibodies.

In another aspect of the present disclosure is a method of detectingmultiple targets in a single tissue sample comprising (i) contacting thetissue sample with a first specific binding entity to detect a firsttarget, the first specific binding entity selected from the groupconsisting of an unmodified antibody, an antibody conjugate, or anepitope-tagged antibody; (ii) contacting the tissue sample with firstdetection reagents to detect the first specific binding entity; (iii)contacting the sample with a second specific binding entity to detect asecond target, the second specific binding entity comprising anepitope-tagged antibody; and (iv) contacting the tissue sample withsecond detection reagents to detect the second specific binding entity,the second detection reagents comprising an anti-tag antibody conjugatedto a detectable moiety.

In some embodiments, method further comprises contacting the sample witha third specific binding entity to detect a third target, the thirdspecific binding entity selected from the group consisting of anunmodified antibody, an antibody conjugate, or an epitope-taggedantibody; and contacting the tissue sample with third detection reagentsto detect the third specific binding entity. In some embodiments, thethird specific binding entity is an epitope-tagged antibody, and whereinthe second and third specific binding entities are introducedsimultaneously.

In some embodiments, the first and third specific binding entities areunmodified antibodies or antibody conjugates, wherein the first andthird specific binding entities are introduced simultaneously. In someembodiments, the introduction of the first and third detection reagentsoccurs before introduction of the second specific binding entity. Insome embodiments, the first and third specific binding entities areunmodified antibodies or antibody conjugates, and wherein the first andthird specific binding entities are introduced sequentially.

In some embodiments, the epitope-tagged antibodies comprise an epitopetag selected from the group consisting of V5, HA, VSV, AU1, AU5, OLLAS,E, E2, KT3, AU1, and OLLAS. In some embodiments, the epitope-taggedantibodies comprise an epitope tag having an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, and SEQ ID NO: 9. In some embodiments, the epitope-taggedantibodies comprise between 4 and 10 epitope tags. In some embodiments,the anti-tag antibodies are conjugated to a fluorescent moiety. In someembodiments, the fluorescent moiety is selected from the groupconsisting of fluorescent moiety selected from xanthene derivatives,cyanine derivatives, squaraine derivatives, naphthalene derivatives,coumarin derivatives, oxadiazole derivatives, anthracene derivatives,pyrene derivatives, oxazine derivatives, acridine derivatives,arylmethine derivatives, and tetrapyrrole derivatives. In someembodiments, at least one of the first, second, or third specificbinding entities is specific for a target selected from the groupconsisting of CD3, CD8, CD20, CD68, PDL1, FoxP3, HER2, and EGFR2.

In another aspect of the present disclosure is an epitope tag constructcomprising tandem repeat epitope tags separated by spacers, the epitopetag construct comprising from 2 to 6 epitope tags. In some embodiments,the epitope tag constructs comprise an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQID NO: 9. In some embodiments, the epitope tag constructs comprise anamino acid sequence having at least 90% identity of a sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,and SEQ ID NO: 9. In some embodiments, at least one of the spacersseparating the epitope tags of the epitope tag construct comprises anamino acid sequence selected from the group consisting of SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. In someembodiments, at least one of the spacers separating the epitope tags ofthe epitope tag construct comprises an amino acid sequence having atleast 90% identity to a sequence selected from the group consisting ofSEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ IDNO: 14. In some embodiments, a molecular weight of the epitope tagconstruct ranges from between about 5 g/mol to about 35 g/mol. In someembodiments, a molecular weight of the epitope tag construct ranges frombetween about 5 g/mol to about 25 g/mol. In some embodiments, theepitope-tag construct comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, epitope tag construct comprises the amino acidsequence of SEQ ID NO: 18. In some embodiments, epitope tag constructcomprises the amino acid sequence of SEQ ID NO: 20. In some embodiments,epitope tag construct comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, epitope tag construct comprises the amino acidsequence of SEQ ID NO: 24. In some embodiments, epitope tag constructcomprises the amino acid sequence of SEQ ID NO: 26. In some embodiments,epitope tag construct comprises the amino acid sequence of SEQ ID NO:28. In some embodiments, epitope tag construct comprises the amino acidsequence of SEQ ID NO: 30. In some embodiments, epitope tag constructcomprises the amino acid sequence of SEQ ID NO: 32. In some embodiments,the epitope-tag construct comprises an amino acid sequence having atleast 90% identity to that of SEQ ID NO: 16. In some embodiments,epitope tag construct comprises an amino acid sequence having at least90% identity to that of SEQ ID NO: 18. In some embodiments, epitope tagconstruct comprises an amino acid sequence having at least 90% identityto that of SEQ ID NO: 20. In some embodiments, epitope tag constructcomprises an amino acid sequence having at least 90% identity to that ofSEQ ID NO: 22. In some embodiments, epitope tag construct comprises anamino acid sequence having at least 90% identity to that of SEQ ID NO:24. In some embodiments, epitope tag construct comprises an amino acidsequence in having at least 90% identity to that of SEQ ID NO: 26. Insome embodiments, epitope tag construct comprises an amino acid sequencehaving at least 90% identity to that of SEQ ID NO: 23. In someembodiments, epitope tag construct comprises on amino acid sequencehaving at least 90% identity to that of SEQ ID NO: 30. In someembodiments, epitope tag construct comprises an amino acid sequencehaving at least 90% identity to that of SEQ ID NO: 32.

In another aspect of the present disclosure is an epitope tag genecomprising a nucleic acid sequence selected from the group consisting of(i) a nucleic acid sequence of SEQ ID NO: 15, (ii) a nucleic acidsequence having at least 90% identity to that of SEQ ID NO: 15, (iii) anucleic acid sequence having at least 95% identity to that of SEQ ID NO:15, and (iv) a nucleic acid sequence having at least 97% identity tothat of SEQ ID NO: 15.

In another aspect of the present disclosure is an epitope tag genecomprising a nucleic acid sequence selected from the group consisting of(i) a nucleic acid sequence of SEQ ID NO: 17, (ii) a nucleic acidsequence having at least 90% identity to that of SEQ ID NO: 17, (iii) anucleic acid sequence having at least 95% identity to that of SEQ ID NO:17, and (iv) a nucleic acid sequence having at least 97% identity tothat of SEQ ID NO: 19.

In another aspect of the present disclosure is an epitope tag genecomprising a nucleic acid sequence selected from the group consisting of(i) a nucleic acid sequence of SEQ ID NO: 19, (ii) a nucleic acidsequence having at least 90% identity to that of SEQ ID NO: 19, (iii) anucleic acid sequence having at least 95% identity to that of SEQ ID NO:19, and (iv) a nucleic acid sequence having at least 97% identity tothat of SEQ ID NO: 19.

In another aspect of the present disclosure is an epitope tag genecomprising a nucleic acid sequence selected from the group consisting of(i) a nucleic acid sequence of SEQ ID NO: 21, (ii) a nucleic acidsequence having at least 90% identity to that of SEQ ID NO: 21, (iii) anucleic acid sequence having at least 95% identity to that of SEQ ID NO:21, and (iv) a nucleic acid sequence having at least 97% identity tothat of SEQ ID NO: 21.

In another aspect of the present disclosure is an epitope tag genecomprising a nucleic acid sequence selected from the group consisting of(i) a nucleic acid sequence of SEQ ID NO: 23, (ii) a nucleic acidsequence having at least 90% identity to that of SEQ ID NO: 23, (iii) anucleic acid sequence having at least 95% identity to that of SEQ ID NO:239, and (iv) a nucleic acid sequence having at least 97% identity tothat of SEQ ID NO: 23.

In another aspect of the present disclosure is an epitope tag genecomprising a nucleic acid sequence selected from the group consisting of(i) a nucleic acid sequence of SEQ ID NO: 25, (ii) a nucleic acidsequence having at least 90% identity to that of SEQ ID NO: 25, (iii) anucleic acid sequence having at least 95% identity to that of SEQ ID NO:25, and (iv) a nucleic acid sequence having at least 97% identity tothat of SEQ ID NO: 25.

In another aspect of the present disclosure is an epitope tag genecomprising a nucleic acid sequence selected from the group consisting of(i) a nucleic acid sequence of SEQ ID NO: 27, (ii) a nucleic acidsequence having at least 90% identity to that of SEQ ID NO: 27, (iii) anucleic acid sequence having at least 95% identity to that of SEQ ID NO:7, and (iv) a nucleic acid sequence having at least 97% identity to thatof SEQ ID NO: 27.

In another aspect of the present disclosure is an epitope tag genecomprising a nucleic acid sequence selected from the group consisting of(i) a nucleic acid sequence of SEQ ID NO: 29, (ii) a nucleic acidsequence having at least 90% identity to that of SEQ ID NO: 29, (iii) anucleic acid sequence having at least 95% identity to that of SEQ ID NO:29, and (iv) a nucleic acid sequence having at least 97% identity tothat of SEQ ID NO: 29.

In another aspect of the present disclosure is an epitope tag genecomprising a nucleic acid sequence selected from the group consisting of(i) a nucleic acid sequence of SEQ ID NO: 31, (ii) a nucleic acidsequence having at least 90% identity to that of SEQ ID NO: 31, (iii) anucleic acid sequence having at least 95% identity to that of SEQ ID NO:31, and (iv) a nucleic acid sequence having at least 97% identity tothat of SEQ ID NO: 31.

In another aspect of the present disclosure is a panel comprising two ormore antibodies, where at least one of the antibodies is anepitope-tagged antibody. In some embodiments, the panel comprises anepitope-tagged antibody selected from the group consisting of anepitope-tagged antibody specific for FoxP3, an epitope-tagged antibodyspecific for CD20, an epitope-tagged antibody specific for CD68, anepitope-tagged antibody specific for FoxP3, an epitope-tagged antibodyspecific for pan-CK, an epitope-tagged antibody specific for PDL1, orany combination thereof. In another aspect of the present disclosure isa panel comprising an epitope-tagged antibody specific for FoxP3, anepitope-tagged antibody specific for CD20, and an epitope-taggedantibody specific for CD68. In some embodiments, the panel furthercomprises an additional specific binding entity. In another aspect ofthe present disclosure is a panel comprising an epitope-tagged antibodyspecific for FoxP3, an epitope-tagged antibody specific for CD8, and anepitope-tagged antibody specific for CD68. In some embodiments, thepanel further comprises an additional specific binding entity. Inanother aspect of the present disclosure is a panel comprising anepitope-tagged antibody specific for FoxP3, an epitope-tagged antibodyspecific for CD8, an epitope-tagged antibody specific for CD68, and anunmodified antibody specific to pan-CK. In some embodiments, the panelfurther comprises an additional specific binding entity. In anotheraspect of the present disclosure is a panel comprising an epitope-taggedantibody specific for FoxP3, an epitope-tagged antibody specific torCD20, an epitope-tagged antibody specific for CD68, and an unmodifiedantibody specific to pan-CK. In some embodiments, the panel furthercomprises an additional specific binding entity. In another aspect ofthe present disclosure is a panel comprising an epitope-tagged antibodyspecific to CD20, an epitope-tagged antibody specific for CD68, anunmodified antibody specific for pan-CK, and an unmodified antibodyspecific for PDL1. In another aspect of the present disclosure is apanel comprising an epitope-tagged antibody specific to CD20, anepitope-tagged antibody specific for CD68, an unmodified antibodyspecific for pan-CK, and an unmodified antibody specific for CD3. Insome embodiments, the panel further comprises an additional specificbinding entity. In another aspect of the present disclosure is a panelcomprising an epitope-tagged antibody specific for FoxP3, anepitope-tagged antibody specific for CD8, an epitope-tagged antibodyspecific for CD68, an epitope-tagged antibody specific for CD3, and anepitope-tagged antibody specific for CD20. In some embodiments, thepanel further comprises an additional specific binding entity.

In another aspect of the present disclosure are kits comprising any ofthe aforementioned panels (assays), where the kits may further comprisedetection reagents for detecting the epitope-tagged antibodies of thepanel (assay).

In another aspect of the present disclosure is an epitope-taggedantibody comprising an antibody and at least one epitope tag construct,the epitope tag construct comprising alternating spacers and epitopetags and having the general structure -[Spacer]a-[Epitope Tag]b, where aand b are each an integer ranging from 1 to 10, and wherein a distancebetween successive -[Epitope Tags]- of the at least one epitope tagconstruct is from 8 to 18 nm. In an embodiment, each epitope tag has aneven number of epitopes. By way of example only, the distance may bemeasured by a contour length, i.e. the linear length of the peptidebackbone. By way of another example only, the distance can be a linearlength between the epitope tags, e.g. the epitope tags in the tertiarystructure. In some embodiments, the distance is less than 12 nm. In someembodiments, the distance is less than 10 nm. In some embodiments, thedistance is less than 9 nm. In some embodiments, the distance isoptimized such that it facilitates bivalent binding of anti-tagantibodies between Epitope Tags. In some embodiments, the distancefacilitates bivalent binding between adjacent epitope tags. In otherembodiments, the distance facilitates bivalent binding betweennon-adjacent epitope tags. In some embodiments, a portion of a firstheavy chain of the anti-tag antibody binds to a first Epitope tag of theepitope tag construct and wherein a portion of a second heavy chain ofthe same anti-tag antibody binds to a second, adjacent Epitope Tag ofthe epitope tag construct. In some embodiments, a portion of a firstheavy chain of the anti-tag antibody binds to a first Epitope Tag of theepitope tag construct and wherein a portion of a second heavy chain ofthe same anti-tag antibody binds to a second, non-adjacent Epitope Tagof the epitope tag construct. In some embodiments, a distance betweencontiguous epitopes is such that the epitopes are accessible by botharms of an anti-tag antibody with elbow angles between about 120 degreesand about 220 degrees, or about 120 degrees to about 200 degrees, orabout 140 degrees to about 200 degrees. In some embodiments, a distancebetween non-contiguous epitopes is such that the epitopes are accessibleby both arms of an anti-tag antibody with elbow angles between about 120degrees and about 220 degrees, or about 120 degrees to about 200degrees, or about 140 degrees to about 200 degrees.

In another aspect of the present disclosure is an epitope-taggedantibody comprising an antibody and at least one epitope tag construct,the epitope tag construct comprising alternating spacers and epitopetags and having the general structure -[Spacer]a-[Epitope Tag]b-, wherea and b are each an integer ranging from 1 to 10, and wherein the-[Spacer]- has a size (e.g. the sum of all atom lengths and bondlengths) which is from 8 to 18 nm in length. In some embodiments, thesize is less than 12 nm. In some embodiments, the -[Spacer]- is sized tofacilitate bivalent binding of anti-tag antibodies between Epitope Tags.In some embodiments, any two adjacent Epitope Tags are spaced a distanceapart from one another which approximates the distance between antigenbinding sites of an anti-tag antibody. In some embodiments, any twonon-adjacent Epitope Tags are spaced a distance apart from one anotherwhich approximates the distance between antigen binding sites of ananti-tag antibody. In some embodiments, a first antigen binding sitebinds to a first Epitope Tag of the at least one epitope tag constructand wherein a second antigen binding site binds to a second, adjacentEpitope Tag of the at least one epitope tag construct. In someembodiments, a first antigen binding site binds to a first Epitope Tagof the at least one epitope tag construct and wherein a second antigenbinding site binds to a second, non-adjacent Epitope Tag of the at leastone epitope tag construct. In some embodiments, a spacer length betweencontiguous epitopes is such that the contiguous epitopes are accessibleby both arms of an anti-tag antibody with elbow angles between about 120degrees and about 220 degrees, or about 120 degrees to about 200degrees, or about 140 degrees to about 200 degrees. In some embodiments,a spacer length is chosen that places non-contiguous epitopes in aconfiguration that the epitopes are accessible by both arms of ananti-tag antibody with elbow angles between about 120 degrees and about220 degrees, or about 120 degrees to about 200 degrees, or about 140degrees to about 200 degrees.

In another aspect of the present disclosure is a system comprising anepitope-tagged antibody as described herein; and a set of detectionreagents for depositing a dye in proximity to each epitope-taggedantibody when the epitope-tagged antibody is bound to a tissue sample,the set of detection reagents comprising an anti-tag specific detectionagent specific for the epitope tag of epitope-tagged antibody. In someembodiments, the anti-tag specific detection agent is conjugated to adye. In some embodiments, the set of detection reagents furthercomprises a first enzyme and a substrate reactive with the first enzymeto deposit the dye on the tissue sample in proximity to the firstenzyme. In some embodiments, the anti-tag specific binding agent isconjugated to the enzyme.

In some embodiments, (i) the anti-tag specific binding agent isconjugated to a hapten, and (ii) the first enzyme is conjugated to anantibody reactive with the hapten. In some embodiments, the anti-tagspecific binding agent is an anti-tag antibody and the first enzyme isconjugated to an anti-species antibody specific for the Ig species ofthe anti-tag antibody. In some embodiments, the anti-tag specificbinding agent is conjugated to a first member of a first specificbinding pair, and wherein the first enzyme is conjugated to a secondmember of the first specific binding pair. In some embodiments, thefirst member of the first specific binding pair is biotin and the secondmember of the first specific binding pair is a biotin-binding protein.In some embodiments, the biotin-binding protein is selected from thegroup consisting of avidin, an avidin derivative (such as NEUTRAVIDIN),and streptavidin. In some embodiments, wherein the first member of thefirst specific binding pair is a hapten and the second member of thefirst specific binding pair is an anti-hapten antibody. In someembodiments, the first anti-tag specific binding agent is conjugated toa horseradish peroxidase enzyme; the first enzyme is conjugated to afirst member of a first specific binding pair; and the set of detectionreagents further comprises a signaling conjugate, the signalingconjugate comprising a tyramide reactive with the second enzyme, whereinthe tyramide is conjugated to a second member of the first specificbinding pair. In some embodiments, the first member of the firstspecific binding pair is a biotin-binding protein; and the second memberof the first specific binding pair is biotin. In some embodiments, thebiotin-binding protein is selected from the group consisting of avidin,an avidin derivative (such as NEUTRAVIDIN), and streptavidin. In someembodiments, the first member of the first specific binding pair is ahapten and the second member of the first specific binding pair is ananti-hapten antibody. In some embodiments, the first anti-tag specificbinding agent is conjugated to a second enzyme; the first enzyme isconjugated to a first member of a first specific binding pair; and theset of detection reagents further comprises a signaling conjugate, thesignaling conjugate comprising a quinone methide reactive with thesecond enzyme, wherein the quinone methide is conjugated to a secondmember of the first specific binding pair. In some embodiments, thefirst member of the first specific binding pair is a biotin-bindingprotein; and the second member of the first specific binding pair isbiotin. In some embodiments, biotin-binding protein is selected from thegroup consisting of avidin, an avidin derivative (such as NEUTRAVIDIN),and streptavidin. In some embodiments, the first member of the firstspecific binding pair is a hapten and the second member of the firstspecific binding pair is an anti-hapten antibody.

In some embodiments, the above-described system comprises a plurality ofthe epitope-tagged antibodies (described above and herein), each of theplurality of the epitope-tagged antibodies is specific for a differentbiomarker, and wherein the set of detection reagents includes means fordepositing a first dye in proximity to at least one of the pluralityepitope-tagged antibodies and at least a second dye in proximity to atleast another of the plurality epitope-tagged antibodies. In someembodiments, the system further comprises an automated slide stainer. Insome embodiments, the system further comprises a slide scanner. In someembodiments, the system further comprises an image analysis system. Insome embodiments, the system further comprises a laboratory informationsystem (LIS). In some embodiments, the LIS comprises a database storingprocessing steps performed on one or more tissue sections of a sample,and/or processing steps to be performed on one or more tissue sectionsof the sample. In some embodiments, the LIS further comprises a set ofinstructions directing the automated slide stainer to deposit theepitope-tagged antibody or the plurality of epitope-tagged antibodies,and the detection reagents on the one or more sections of a tissuesample.

Applicants have discovered that the epitope-tagged antibodies accordingto the present disclosure avoid cross-reactivity often observed whensame-species primary antibodies are used in multiplex assays. As aresult, multiple epitope-tagged primary antibodies may be pooledtogether as a single reagent in a multiplex assay. Moreover, Applicantshave found that the epitope-tagged antibodies of the present disclosureare more stable than chemically modified antibodies, such as thoseantibodies chemically conjugated to a hapten, and are able to bemanufactured consistently. In addition, Applicants have found that theepitope-tagged antibodies provide a linear signal detection cascade thatallows for direct quantitative correlation of protein expression levels,in comparison to detection using signal amplification methods, e.g.tyramide signal amplification. As such, Applicants submit that a MIHCassay employing a plurality of epitope-tagged antibodies, alone or inconjunction with unmodified antibodies, antibody conjugates or otherspecific binding entities, effectively allows for the detection ofmultiple targets in a tissue sample, where the MIHC assay may becompleted in less than five hours, and where the MIHC allows for signalco-localization.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided to the Office upon request and thepayment of the necessary fee.

FIG. 1 illustrates a multiplex IHC assay utilizing three differentepitope-tagged antibodies.

FIGS. 2A through 2D are images of tissue samples stained according to amultiplex IHC assay utilizing three different epitope-tagged antibodies.

FIG. 3 illustrates a multiplex IHC assay utilizing three differentepitope-tagged antibodies.

FIGS. 4A and 4B are images of tissue samples stained according to amultiplex IHC assay utilizing three different epitope-tagged antibodies.

FIG. 5 illustrates a multiplex IHC assay utilizing one unmodifiedantibody and three different epitope-tagged antibodies.

FIGS. 6A through 6F are images of tissue samples stained according to amultiplex IHC assay utilizing one unmodified antibody and threedifferent epitope-tagged antibodies.

FIG. 7 illustrates a multiplex IHC assay utilizing one unmodifiedantibody and three different epitope-tagged antibodies.

FIGS. 8A through 8B are images of tissue samples stained according to amultiplex IHC assay utilizing one unmodified antibody and threedifferent epitope-tagged antibodies.

FIG. 9 illustrates a multiplex IHC assay utilizing two unmodifiedantibodies and two different epitope-tagged antibodies.

FIGS. 10A through 10G are images of tissue samples stained according toa multiplex IHC assay utilizing two unmodified antibodies and twodifferent epitope-tagged antibodies.

FIG. 11 illustrates a multiplex IHC assay utilizing two unmodifiedantibodies and two different epitope-tagged antibodies.

FIGS. 12A through 12D are images of tissue samples stained according toa multiplex IHC assay utilizing two unmodified antibodies and twodifferent epitope-tagged antibodies.

FIG. 13 illustrates a multiplex IHC assay utilizing five differentepitope-tagged antibodies.

FIGS. 14A through 14J are images of tissue samples stained according toa multiplex IHC assay utilizing five different epitope-taggedantibodies.

FIGS. 15A, 15B, and 15C provide flowcharts illustrating various methodsof conducting multiplex IHC assays utilizing one or more epitope-taggedantibodies and/or one or more unmodified antibodies to antibodyconjugates.

FIGS. 16A through 16E provide absorbance data corresponding toepitope-tagged antibodies as compared with their corresponding nativeantibodies (unmodified antibodies).

FIGS. 17A through 17F provide images showing the comparative stainingperformances of epitope-tagged antibodies as compared with correspondingnative antibodies (unmodified antibodies).

FIG. 18 illustrates the production yield of epitope-tagged antibodies ascompared with corresponding native antibodies.

FIG. 19 illustrates the stability of epitope-tagged antibodies, thestability determined by accelerated stability testing studies; TheArrhenius model was followed to predict real-time stability of testedantibodies (1 ug/ml concentration in diluent 90103) at intended storageconditions (i.e. 4° C.) based an data collected at elevated temperatures(i.e. 37° C. and 45° C.) for shorter periods (e.g. 5 to 10 days).

FIGS. 20A through 20F illustrate the structure of an epitope tagconstruct and the spatial and structural relationships between theepitope tags and spacers comprising the epitope tag construct.

FIG. 21 sets forth a step-wise assay procedure.

FIG. 22 sets forth a step-wise assay procedure.

FIG. 23 sets forth a step-wise assay procedure.

FIG. 24A provides kinetic data from a BLI assay indicating that theantibodies with four tags on both the heavy and light chains dissociateat a much slower rate than the same antibody with four tags only on theheavy chain or only on the light chain.

FIG. 24B provides an avidity effect comparison illustrating theadvantage of antibodies with four tags on both the heavy and lightchains.

FIG. 24C illustrates MsAntiV5 recognition with HER2 H0KX, where lightchain tags show a similar BLI response to heavy chain tags; MsAntiV5showed faster on-rate and similar BLI response to GtAntiRb with H0K3;and the the figure further illustrates that BLI response becamesaturated with approximately two to three V5 tags (which matched tissuestaining results). There was also an observed decreased anti-V5dissociation with higher V5-tag loading.

FIG. 25A illustrates the structure of certain epitope-tagged antibodiesused for DLS/DSC testing.

FIG. 25B shows that BSA was quantitatively removed from the samples (SDSpage).

FIG. 25C provides DLS measurement data;

FIGS. 25D and 25E illustrate various DLS temperature ramps; and

FIG. 25F illustrates the results of a DLS analysis.

FIG. 26 sets forth dynamic light scattering data for several differentepitope-tagged antibodies and their respective untagged antibody.

FIGS. 27A through 27C set forth SEC data for various epitope-taggedantibodies and their respective untagged antibody.

FIGS. 28A through 28E set forth DSC data for various epitope-taggedantibodies and their respective untagged antibody.

FIGS. 29A through 29E set forth DLS data for various epitope-taggedantibodies and their respective untagged antibody.

FIG. 30A through 30C provide a schematic illustration of the basic SPRexperiment for measuring the binding of an analyte molecule to areceptor molecule.

FIG. 31A through 31E illustrate kinetic assessments of the bindingbehavior of rabbit monoclonal antibodies towards singly chemicallybiotinylated peptidic 2 kDa analytes. Mab<V5>rRb-J53_wt andMab<V5>rRb-J53_H1L2 show a 1:1 binding stoichiometry. There were nokinetic differences observable between peptide analytes with biotinposition in bi-1 or bi-14 (FIG. 31A). Mab<E>rRb-J26_wt andMab<E>rRb-J26_H2L5 show three digit nanomolar affinities and 1:2 bindingstoichiometry MR=2 (FIG. 31B). Mab<E2>rRb-J78_wt and Mab<E2>rRb-J78_H5Lshow two to three digit nanomolar affinities and 1:1 bindingstoichiometry MR=1. Peptides with biotin in position bi-1 wererecognized with higher affinity than with biotinylation in positionbi-12 (FIG. 31C). Mab<HA>rRb-J15_H2L2 showed affinity in the three digitnanomolar range with 1:1 binding stoichiometry. No kinetic differencewas observed between peptide analytes with biotin at position bi-1 or atposition bi-9 (FIG. 31D). Mab<VSV-G>rRb-J110_H5L2 shows three digitnanomolar affinity and 1:2 binding stoichiometry (FIG. 31E).

FIG. 32A through 32D illustrate kinetic assessments of the bindingbehavior of rabbit monoclonal anti-tag antibodies towards the respectivetagged antibody analytes: CD68 HLvsv, CD68 HvsvLvsv, CD68 HvsvL, CD68 HL(FIG. 32A); CD8 HL, CD8 He2L (FIG. 32B top); PD-L1 SP63 He2L, PD-L1 SP63HLe2, PD-L1 SP63 He2Le2, PD-L1 SP63 HL (FIG. 32B bottom); CD20 HL, CD20HhaL, CD20 HLha, CD20 HhaLha (FIG. 32C top); PD-L1 SP63 HhaL, PD-L1 SP63HLha, PD-L1 SP63 HhaLha, PD-L1 SP63 HL (FIG. 32C bottom); FoxP3 HL,FoxP3 H5v5L, FoxP3 HL5v5, FoxP3 H5v5L5v5, FoxP3 H4v5L, FoxP3 HL4v5,FoxP3 H4v5L4v5 (FIG. 32D).

FIG. 33A (A) Schematic representation of recombinant epitope-taggedantibody and (B) detection strategy of 5-plex fluorescent multiplexdetection without successive removal of serially deposited primaryantibodies and anti-species antibody conjugates. In addition toenzyme-mediated fluorophore deposition, an additional signalamplification configuration involves using hapten-conjugated anti-tagand fluorophore-conjugated anti-hapten antibodies as described in (C).

FIG. 33B provides a FFPE NSCLC section probed with epitope-taggedantibodies against PD-L1 (cyan), CD3 (gold), CD8 (green), FoxP3 (red)and CD68 (purple). The markers were detected by fluorophore-conjugatedanti-tag antibodies and antibody-enzyme conjugate-mediated deposition offluorophores onto tissue components as described in FIG. 1. Slides werescanned using Zeiss Axio Scan.ZI equipped with six cube filters andvisualized in Zen software following pseudo-coloring. Colors wereassigned based on fluorophore emission wavelength in the color spectrumwith exception of CD68. Cell nuclei were marked by DAPI stain (Blue).The salmon colored objects are red blood cells (RBC) that auto-fluorescein multiple channels.

FIG. 33C provides FFPE lung tumor sections probed with (A) tagged and(B) native anti-CD8 antibodies, (C) tagged and (D) native anti-FoxP3antibodies, and (F) tagged and (F) native anti-CD68 antibodies. Themarkers were detected using (A, C, and E) fluorophore-conjugatedanti-tag antibodies or (B, D, and F) VENTANA ultraView Universal DABDetection Kit. Fluorescently stained slides were co-stained with VENTANADISCOVERY QD DAPI.

FIG. 33D provides a FFPE lung tumor sections probed with (A) tagged and(B) native anti-PD-L1(SP263) antibodies or with (C) tagged and (D)native anti-CD3 antibodies. The markers were detected using (A and C)enzyme-conjugated anti-tag antibodies and QM- and TSA-fluor deposition,(B) VENTANA OptiView DAB IHC, or (D) VENTANA ultraView Universal DABdetection kits.

FIG. 33E provides a boxplot of 10 NSCLC cases showing coverage of eachmarker stained using 5-plex fluorescent assay compared against DAB. Themarkers were detected as described in FIGS. 33A through 33D. Scannedfluorescent and DAB whole slide images were evaluated by pathologistafter brightness adjustment and without further processing. The fewcases that exhibited discordance between PD-L1 and CD8 fluorescence andDAB staining had unusually high autofluorescence associated withextensive connective tissue in the lung. Connective autofluorescencecould be removed digitally using signal unmixing procedures.

FIG. 34A sets forth size exclusion chromatography elution profiles of0.5 μM anti-PD-L1-E2 (H4K0), 0.5 μM anti-E2, and anti-E2 mixed with 0.5μM anti-PD-L1-E2 at varying molar ratios from 0.5:1 to 6:1.

FIG. 34B sets forth size exclusion chromatography elution profiles of0.5 μM anti-CD8-E2 (H4K0), 0.5 μM anti-E2, and anti-E2 mixed with 0.5 μManti-CD8-E2 at varying molar ratios from 0.5:1 to 6:1.

FIG. 34C sets forth size exclusion chromatography elution profiles of0.5 μM anti-CD3-E (H4K0), 0.5 μM anti-E, and anti-E mixed with 0.5 μManti-CD3-E at varying molar ratios from 0.5:1 to 6:1.

FIG. 34D sets forth size exclusion chromatography elution profiles of0.5 μM anti-FoxP3-V5 (H4K0), 0.5 μM anti-V5, and anti-V5 mixed with 0.5μM anti-FoxP3-V5 at varying molar ratios from 0.5:1 to 6:1.

FIG. 34E sets forth size exclusion chromatography elution profiles of0.5 μM anti-CD68-VSV-G (H4K0), 0.5 μM anti-VSV-G, and anti-VSV-G mixedwith 0.5 μM anti-C68-VSV-G at varying molar ratios from 0.5:1 to 6:1.

FIG. 34F sets forth size exclusion chromatography elution profiles of0.5 μM anti-CD8-AU5 (H4K0), 0.5 μM anti-AU5, and anti-AU5 mixed with 0.5μM anti-CD8-AU5 at varying molar ratios from 0.5:1 to 6:1.

FIG. 34G sets forth size exclusion chromatography elution profiles of0.5 μM anti-FoxP3-V5 (H5K0), 0.5 μM anti-V5, and anti-V5 mixed with 0.5μM anti-FoxP3-V5 at varying molar ratios from 0.5:1 to 8:1.

FIG. 35A illustrates the bivalent binding of an anti-tag antibody to theexpressed epitope tags of an epitope-tagged antibody, wherein bindingoccurs between adjacent epitope tags.

FIG. 35B illustrates the bivalent binding of an anti-tag antibody to theexpressed epitope tags or an epitope-tagged antibody, wherein bindoccurs between non-adjacent epitope tags.

DETAILED DESCRIPTION

In general, the present disclosure is directed to epitope-taggedantibodies, as well as methods of employing the epitope-taggedantibodies for detecting one or more targets in a biological sample,e.g. a tissue sample.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless the context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise.

As used herein, the terms “comprising,” “including,” “having,” and thelike are used interchangeably and have the same meaning. Similarly,“comprises,” “includes,” “has,” and the like are used interchangeablyand have the same meaning. Specifically, each of the terms is definedconsistent with the common United States patent law definition of“comprising” and is therefore interpreted to be an open term meaning “atleast the following,” and is also interpreted not to exclude additionalfeatures, limitations, aspects, etc. Thus, for example, “a device havingcomponents a, b, and c” means that the device includes at leastcomponents a, b and c. Similarly, the phrase: “a method involving stepsa, b, and c” means that the method includes at least steps a, b, and c.Moreover, while the steps and processes may be outlined herein in aparticular order, the skilled artisan will recognize that the orderingsteps and processes may vary.

As used herein, the term “affinity” refers to the non-random interactionof two molecules. The term “affinity” refers to the strength ofinteractions and can be expressed quantitatively as a dissociationconstant (KD). One or both of the two molecules may be a peptide (e.g.antibody). Binding affinity (i.e., KD) can be determined using standardtechniques. For example, the affinity can be a measure of the strengthof the binding of an individual epitope with an antibody molecule.

As used herein, the term “antibody,” refers to immunoglobulins orimmunoglobulin-like molecules, including by way of example and withoutlimitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, andsimilar molecules produced during an immune response in any vertebrate,(e.g., in mammals such as humans, goats, rabbits and mice) and antibodyfragments (such as F(ab′)2 fragments, Fab′ fragments, Fab′-SH fragmentsand Fab fragments as are known in the art, recombinant antibodyfragments (such as sFv fragments, dsFv fragments, bispecific sFvfragments, bispecific dsFv fragments, F(ab)′2 fragments, single chain Fvproteins (“scFv”), disulfide stabilized Fv proteins (“dsFv”), diabodies,and triabodies (as are known in the art), and camelid antibodies) thatspecifically bind to a molecule of interest (or a group of highlysimilar molecules of interest) to the substantial exclusion of bindingto other molecules. Antibody further refers to a polypeptide ligandcomprising at least a light chain or heavy chain immunoglobulin variableregion which specifically recognizes and binds an epitope of an antigen.Antibodies may be composed of a heavy and a light chain, each of whichhas a variable region termed the variable heavy (VH) region and thevariable light (VL) region. Together, the VH region and the VL regionare responsible for binding the antigen recognized by the antibody. Theterm antibody also includes intact immunoglobulins and the variants andportions of them well known in the art.

As used herein, the term “antigen” refers to a compound, composition, orsubstance that may be specifically bound by the products of specifichumoral or cellular immunity, such as an antibody molecule or T-cellreceptor. Antigens can be any type of molecule including, for example,haptens, simple intermediary metabolites, sugars (e.g.,oligosaccharides), lipids, and hormones as well as macromolecules suchas complex carbohydrates (e.g., polysaccharides), phospholipids, nucleicacids and proteins.

As used herein, the term “avidity” refers to the cooperative andsynergistic bonding of two or more molecules. “Avidity” refers to theoverall stability of the complex between two or more populations ofmolecules, that is, the functional combining strength of an interaction.

As used herein, “biological sample” or “tissue sample” can be any solidor fluid sample obtained from, excreted by or secreted by any livingorganism, including without limitation, single celled organisms, such asbacteria, yeast, protozoans, and amoebas among others, multicellularorganisms (such as plants or animals, including samples from a healthyor apparently healthy human subject or a human patient affected by acondition or disease to be diagnosed or investigated, such as cancer).For example, a biological sample can be a biological fluid obtainedfrom, for example, blood, plasma, serum, urine, bile, ascites, saliva,cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion,a transudate, an exudate (for example, fluid obtained from an abscess orany other site of infection or inflammation), or fluid obtained from ajoint (for example a normal joint or a joint affected by disease). Abiological sample can also be a sample obtained from any organ or tissue(including a biopsy or autopsy specimen, such as a tumor biopsy) or caninclude a cell (whether a primary cell or cultured cell) or mediumconditioned by any cell, tissue or organ. The samples may be tumorsamples, including those from melanoma, renal cell carcinoma, andnon-small-cell lung cancers. In some embodiments, the samples areanalyzed for the of cancer by detecting targets, including biomarkers(e.g. proteins or nucleic acid sequences), within the tissue sample. Thedescribed embodiments of the disclosed method can also be applied tosamples that do not have abnormalities, diseases, disorders, etc.referred to as “normal” samples or “control” samples. For example, itmay be useful to test a subject for cancer by taking tissue samples frommultiple locations, and these samples may be used as controls andcompared to later samples to determine whether a particular cancer hasspread beyond its primary origin.

As used herein, “conjugate” refers to two or more molecules (and/ormaterials such as nanoparticles) that are covalently linked into alarger construct. In some embodiments, a conjugate includes one or morebiomolecules (such as peptides, proteins, enzymes, sugars,polysaccharides, lipids, glycoproteins, and lipoproteins) covalentlylinked to one or more other molecules, such as one or more otherbiomolecules. In other embodiments, a conjugate includes one or morespecific-binding molecules (such as antibodies) covalently linked to oneor more detectable labels (such as a fluorophore, a luminophore,fluorescent nanoparticles, haptens, enzymes and combinations thereof).

As used herein, “detection probes” include nucleic acid probes orprimary antibodies which bind to specific targets (e.g. nucleic acidsequences, proteins, etc.). The detection probes may include a label fordirect detection, such as radioactive isotopes, enzyme substrates,co-factors, ligands, chemiluminescent or fluorescent agents, haptens(including, but not limited to, DNP), and enzymes. Alternatively, thedetection probes may contain no label or tag and may be detectedindirectly (e.g. with a secondary antibody that is specific for thedetection probe).

As used herein, “haptens” are small molecules that can combinespecifically with an antibody, but typically are substantially incapableof being immunogenic except in combination with a carrier molecule. Insome embodiments embodiments, haptens include, but are not limited to,pyrazoles (e.g. nitropyrazoles); nitrophenyl compounds; benzofurazans;triterpenes; ureas (e.g. phenyl ureas); thioureas (e.g. phenylthioureas); rotenone and rotenone derivatives; oxazole (e.g. oxazolesulfonamides); thiazoles (e.g. thiazole sulfonamides); coumarin andcoumarin derivatives; and cyclolignans. Additional non-limiting examplesof haptens include thiazoles; nitroaryls; benzofurans; triperpenes; andcyclolignans. Specific examples of haptens include di-nitrophenyl,biotin, digoxigenin, and fluorescein, and any derivatives or analogsthereof. Other haptens are described in U.S. Pat. Nos. 8,846,320;8,618,265; 7,695,929; 8,481,270; and 9,017,954, the disclosures of whichare incorporated herein by reference in their entirety. The haptensthemselves may be suitable for direct detection, i.e. they may give offa suitable signal for detection.

As used herein, the term “hydrodynamic radius,” as used herein, denotesthe radius of a sphere having equivalent hydrodynamic properties as agiven structure. The hydrodynamic radius is derived from the diffusioncoefficient by the Stokes Einstein equation.

As used herein, “immunohistochemistry” refers to a method of determiningthe presence or distribution of an antigen in a sample by detectinginteraction of the antigen with a specific binding agent, such as anantibody. A sample is contacted with an antibody under conditionspermitting antibody-antigen binding. Antibody-antigen binding can bedetected by means of a detectable label conjugated to the antibody(direct detection) or by means of a detectable label conjugated to asecondary, antibody, which binds specifically to the primary antibody(indirect detection).

As used herein, “multiplex,” “multiplexed,” or “multiplexing” refer todetecting multiple targets in a sample concurrently, substantiallysimultaneously, or sequentially. Multiplexing can include identifyingand/or quantifying multiple distinct nucleic acids (e.g., DNA, RNA,mRNA, miRNA) and polypeptides (e.g., proteins) both individually and inany and all combinations.

As used herein, the term “primary antibody” refers to an antibody whichbinds specifically to a target protein antigen in a tissue sample. Aprimary antibody is generally the first antibody used in animmunohistochemical procedure. Epitope-tagged antibodies, unmodifiedantibodies, or antibody conjugates, each described herein, are examplesof primary antibodies. Primary antibodies may thus serve as “detectionprobes” for detecting a target within a tissue sample.

As used herein, the term “secondary antibody” herein refers to anantibody which binds specifically to a detection probe or portionthereof (e.g. a hapten or a primary antibody), thereby forming a bridgebetween the detection probe and a subsequent reagent (e.g. a label, anenzyme, etc.), if any. A secondary antibody may be used to indirectlydetect detection probes, e.g. primary antibodies. Examples of secondaryantibodies include anti-tag antibodies, anti-species antibodies, andanti-label antibodies, each described herein.

As used herein, the term “specific binding entity” refers to a member ofa specific-binding pair. Specific binding pairs are pairs of moleculesthat are characterized in that they bind each other to the substantialexclusion of binding to other molecules (for example, specific bindingpairs can have a binding constant that is at least 103 M-1 greater, 104M-1 greater or 105 M-1 greater than a binding constant for either of thetwo members of the binding pair with other molecules in a biologicalsample). Particular examples of specific binding moieties includespecific binding proteins (for example, antibodies, lectins, avidinssuch as streptavidins, and protein A). Specific binding moieties canalso include the molecules (or portions thereof) that are specificallybound by such specific binding proteins. Specific binding entitiesinclude primary antibodies, described above, or nucleic acid probes.

As used herein, “target” means any molecule for which the presence,location and/or concentration is or can be determined. Examples oftargets include nucleic acid sequences and proteins, such as thosedisclosed herein.

Epitope Tagged Antibodies

In one aspect of the present disclosure are recombinant antibodies thatexpress one or more molecularly engineered epitope tags (“epitope-taggedantibodies”). In general, expression of the epitope tags (short aminoacid sequences, namely antigenic peptide sequences) allows theepitope-tagged antibody to be recognized and/or detected (such as withan anti-tag antibody, described further herein). Thus, the epitopetagged antibodies disclosed herein are suitable for use inimmunohistochemical assays, including in multiplex immunohistochemicalassays, and thereby may be used as primary antibodies or detectionprobes such that targets within a tissue sample may be detected.

An epitope-tagged antibody may be derived from any name antibody. Thus,like native or unmodified antibodies the epitope-tagged antibodies showspecificity for a particular target. For example, an epitope-taggedantibody may be derived from antibodies specific for cluster ofdifferentiation markers (e.g. CD3, CD8, CD20, CD68), HER2, FoxP3, PDL1,and EGFR2. Other non-limiting targets, e.g. protein targets, for whichthe epitope-tagged antibodies may be developed for detection purposesare disclosed further herein.

In general, the epitope-tagged antibodies comprise at least one epitopetag construct, the epitope tag constructing comprising alternatingspacers and epitope tags. As used here, the term “epitope tag construct”refers to alternating epitope tags and spacers, e.g. an amino acidsequence comprising tandem repeat epitope tags separated by spacers(see, for instance, FIG. 20A).

For illustration purposes only, the epitope tagged antibodies of thepresent disclosure may have the structure:

Ab-([Spacer]-[Epitope Tag])_(n)-[Spacer], where n is an integer rangingfrom 1 to 20, “Ab” is an antibody, and “Epitope Tag” and “Spacer” are asdefined herein, and where each of the “Spacers” may be the same ordifferent and where ([Spacer]-[Epitope Tag]) represents an example of anepitope tag construct.

The epitope-tagged antibodies are engineered such that the epitope tagsand spacers are incorporated at the terminal end of a heavy chainconstant region, at the terminal end of a light chain constant region,or at both the terminal ends of a heavy chain constant region and alight chain constant region. In some embodiments, an epitope tagconstruct is comprised at the C-terminal end of a heavy chain constantregion of an antibody, a C-terminal end of light chain constant regionof an antibody, or at both the C-terminal ends of heavy and light chainconstant regions of an antibody.

In other embodiments, an epitope tag construct is comprised solely atthe C-terminal end of the heavy chain constant region of the antibody.In other embodiments, an epitope tag construct is compared solely at theC-terminal end of the light chain constant region of the antibody. Insome embodiments, the heavy chain constant regions are identified hereinby the notation “H;” while the light chain constant regions areidentified herein by the notation “K.” For example, the notation H4K4may refer to an epitope-tagged antibody comprising four epitope tags ata terminal end of a heavy chain constant region and an additional fourepitope tags at a terminal end of a light chain constant region.Likewise, the notation H4K0 may refer to an epitope-tagged antibodycomprising four epitope tags at a terminal end of a heavy chain constantregion, and no epitope tags at a terminal end of a light chain constantregion.

Any type of epitope tag may be incorporated into an antibody, providedthat the epitope tag does not impair the function of the antibody (i.e.impede the ability of the antibody to bind to targets or prevent theantibody from being detected). In some embodiments, an epitope tag andits configuration on IgG (the number of tags, tag's position on IgG) isconsidered during selection such that it minimizes any tertiarystructure disruptions of the antibody to which it is incorporated. Insome embodiments, an epitope tag is selected such that it has nocross-reactivity with human proteins. In some embodiments, the epitopetag has a nucleotide sequence comprising between about 0 and about 20bases. In other embodiments, the epitope tag has a nucleotide sequencecomprising between about 6 and about 16 bases. The sequence for theepitope tag may be derived from a naturally occurring source or may besynthetic.

In some embodiments, the epitope tag is selected from a VSV epitope tag(SEQ ID NO: 1), an AU5 epitope tag (SEQ ID NO: 2), an E epitope tag (SEQID NO: 3), a V5 epitope tag (SEQ ID NO: 4), an HA epitope tag (SEQ IDNO: 5), or an E2 epitope tag (SEQ ID NO: 6). Other suitable epitope tagsthat may be incorporated within epitope-tagged antibodies include FLAG,AU1 (SEQ ID NO:8), OLLAS (SEQ ID NO: 9), and KT3 (SEQ ID NO: 7). Ofcourse, these exemplified epitope tags may be modified according toprocedures known to those of ordinary skill in the art.

The epitope-tagged antibodies (or any epitope tag construct incorporatedinto an epitope-tagged antibody) may comprise or express one epitope tagor a plurality of epitope tags. In general, the epitope taggedantibodies of the present disclosure may comprise any number of epitopetags provided that the epitope tags do not interfere with or reduce thefunction of the antibody. In some embodiments, the epitope faggedantibodies comprise at least 2 epitope, tags. In other embodiments, theepitope tagged antibodies comprise at least 3 epitope tags. In yet otherembodiments, the epitope tagged antibodies comprise at least 4 epitopetags. In further embodiments, the epitope tagged antibodies comprisebetween 2 and 20 epitope tags. In yet further embodiments, the epitopetagged antibodies comprise between 2 and 12 epitope tags. In evenfurther embodiments, the epitope tagged antibodies comprise between 3and 9 epitope tags. In one particular embodiment, the epitope taggedantibodies comprise 4 epitope tags. In another particular embodiment,the epitope tagged antibodies comprise 5 epitope tags.

In some embodiments, any epitope tag construct comprises between 2 and10 epitope tags. In some embodiments, any epitope tag constructcomprises between 2 and 8 epitope tags. In other embodiments, anyepitope tag construct comprises between 2 and 5 epitope tags. In yetother embodiments, any epitope tag construct comprises 4 or 5 epitopetags.

In some embodiments, the number of epitope tags incorporated (or“expressed”) at the C-terminal end of a heavy chain constant region isgreater than the number of epitope tags incorporated at the C-terminalend of a light chain constant region (i.e. the number of epitope tagsconstituting an epitope tag construct incorporated at the C-terminal endof a heavy chain constant region is greater than the number of epitopetags constituting an epitope tag construct incorporated at theC-terminal end of a light chain constant region). In other embodiments,the number of epitope tags incorporated at the C-terminal end of a heavychain constant region is less than the number of epitope tagsincorporated at the C-terminal end of a light chain constant region(i.e. the number of epitope tags constituting an epitope tag constructat the C-terminal end of a heavy chain constant region is less than thenumber of epitope tags constituting an epitope tag construct at theC-terminal end of a light chain constant region).

In some embodiments, a ratio of the number of epitope tags incorporatedat the C-terminal end of a heavy chain constant region to the number ofepitope tags incorporated at the C-terminal end of a light chainconstant region ranges from 4:1 to about 1:4. In other embodiments, aratio of the number of epitope tags incorporated at the C-terminal endof a heavy chain constant region to the number of epitope tagsincorporated at the C-terminal end of a light chain constant region isranges from 2:1 to about 1:2. In yet other embodiments, a ratio of thenumber of epitope tags incorporated at the C-terminal end of a heavychain constant region to the number of epitope tags incorporated at theC-terminal end of a light chain constant region is ranges from 1.5:1 toabout 1:1.5. In further embodiments, a ratio of the number of epitopetags incorporated at the C-terminal end of a heavy chain constant regionto the number of epitope tags incorporated at the C-terminal end of alight chain constant region is about 1:1.

In some embodiments, between 2 and 8 epitope tags are incorporated atthe C-terminal end of a heavy chain constant region and between 2 and 8epitope tags are incorporated at the C-terminal end of a light chainconstant region. In some embodiments, between 2 and 8 epitope tags areincorporated at the C-terminal end of a heavy chain constant region andbetween 0 and 5 epitope tags are incorporated at the C-terminal end of alight chain constant region. In other embodiments, between 2 and 6epitope tags are incorporated at the C-terminal end of a heavy chainconstant region and between 0 and 4 epitope tags are incorporated at theC-terminal end of a light chain constant region. In other embodiments, 4or 5 epitope tags are incorporated at the C-terminal end of a heavychain constant region and between 2 and 5 epitope tags are incorporatedat the C-terminal end of a light chain constant region. In yet otherembodiments, 4 or 5 epitope tags are incorporated at the C-terminal endof a heavy chain constant region and 0, 1, or 2 epitope tags areincorporated at the C-terminal end of a light chain constant region. Infurther embodiments, 4 or 5 epitope tags are incorporated at theC-terminal end of a heavy chain constant region and no epitope tags areincorporated at the C-terminal end of a light chain constant region. Inother embodiments, at least 2 epitope tags are incorporated at theC-terminal end of a light chain constant region and at least 1 epitopetag is incorporated at the C-terminal end of a heavy chain constantregion. In other embodiments, at least 3 epitope tags are incorporatedat the C-terminal end of a light chain constant region and at least 1epitope tag is incorporated at the C-terminal end of a heavy chainconstant region.

In one particular embodiment, the epitope-tagged antibody has theconfiguration H1K0. In another particular embodiment, the epitope-taggedantibody has the configuration H2K0. In another particular embodiment,the epitope-tagged antibody has the configuration H3K0. In anotherparticular embodiment, the epitope-tagged antibody has the configurationH4K0. In another particular embodiment, the epitope-tagged antibody hasthe configuration H5K0. In another particular embodiment, theepitope-tagged antibody has the configuration H0K1. In anotherparticular embodiment, the epitope-tagged antibody has the configurationH0K2. In another particular embodiment, the epitope-tagged antibody hasthe configuration H0K3. In another particular embodiment, theepitope-tagged antibody has the configuration H0K4. In anotherparticular embodiment, the epitope-tagged antibody has the configurationH0K4. In another particular embodiment, the epitope-tagged antibody hasthe configuration H2K2. In another particular embodiment, theepitope-tagged antibody has the configuration H3K3. In anotherparticular embodiment, the epitope-tagged antibody has the configurationH4K4.

As noted herein, the epitope tags are separated from each other by aspacer. In some embodiments, a space is provided at the terminal end ofthe heavy and/or light chain constant region such that any epitope tagconstruct is “coupled” to the spacer rather than directly to theterminal end of the respective constant region. In some embodiments, anadditional sequence (e.g. a linking or coupling sequence) is providedbetween the terminal end of the heavy and/or light chain constantregion, bridging the terminal end to the spacer (or to the epitope-tagconstruct). Likewise, in some embodiments, at least one additionalspacer is provided after a terminal epitope tag of an epitope tagconstruct, but prior to any stop codon sequence. FIGS. 20A through 20Ffurther illustrate the structure of an epitope tag construct and thespatial and structural relationships between epitope tags (outlinedwithin rectangular boxes) and spacers (sequences between the epitopetags). FIGS. 20A through 20F also illustrate restriction sites withinthe “epitope tag gene” (defined herein) and the position of a stopcodon.

The spacer itself is selected such that the amino acid sequence thespacer (or its resulting tertiary structure) of does not interfere withany folding protein domains or the antibody. In some embodiments, thespacer comprises a nucleic acid sequence having between about 20 basesand about 60 bases. In some embodiments, the size of any spacer isselected such that the spacer sufficiently separates epitope tags tofacilitate the binding of multiple anti-tag antibodies to theepitope-tagged antibody.

In some embodiments, the spacers comprise an amino acid sequenceselected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, or SEQ ID NO: 15. In some embodiments, the spacerscomprise an amino acid sequence having at least 75% identity to asequence selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, or SEQ ID NO: 14. In some embodiments, the spacers comprisean amino acid sequence having at least 80% identity to a sequenceselected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, or SEQ ID NO: 14. In some embodiments, the spacers comprise an aminoacid sequence having at least 85% identity to a sequence selected fromSEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ IDNO: 14. In some embodiments, the spacers comprise an amino acid sequencehaving at least 90% identity to a sequence selected from SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. In someembodiments, the spacers comprise an amino acid sequence having at least95% identity to a sequence selected from SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

In some embodiments, at least a portion of the amino acid sequenceconstituting the spacer comprises an amino acid sequence including asequence of SEQ ID NO: 10; SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,or SEQ ID NO: 14. In some embodiments, at least a portion of the aminoacid sequence constituting the spacer comprises an amino acid sequencebasing at least 75% to a sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. In some embodiments, atleast a portion of the amino acid sequence constituting the space;comprises an amino acid sequence including a sequence of SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. In someembodiments, at least a portion of the amino acid sequence constitutingthe spacer comprises an amino acid sequence having at least 80% to asequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,or SEQ ID NO: 14. In some embodiments, at least a portion of the aminoacid sequence constituting the spacer comprises an amino acid sequenceincluding a sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, or SEQ ID NO: 14. In some embodiments, at least a portion ofthe amino acid sequence constituting the spacer comprises an amino acidsequence having at least 85% to a sequence of SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. In some embodiments,at least a portion of the amino acid sequence constituting the spacercomprises an amino acid sequence including a sequence of SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. In someembodiments, at least a portion of the amino acid sequence constitutingthe spacer comprises an amino acid sequence having at least 90% to asequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,or SEQ ID NO: 14. In some embodiments, at least a portion of the aminoacid sequence constituting the spacer comprises an amino acid sequenceincluding a sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, or SEQ ID NO: 14. In some embodiments, at least a portion ofthe amino acid sequence constituting the spacer comprises an amino acidsequence having at least 95% to a sequence of SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

The spacers separating the epitope tags may be the same or different.For example, if an epitope tag construct comprises five epitope tags,the spacers between the five epitope tags of the construct may be sameor different. In some embodiments, the spacers separating the epitopetags of any epitope tag construct are all the same. In otherembodiments, the spacers separating the epitope tags of any epitope tagconstruct are all different from one another. In yet other embodiments,at least some of the spacers separating the epitope tags of any epitopetag construct are the same. In some embodiments, a portion of any spacermay be the same or different than a portion of another spacer. In someembodiments, a portion of any sequence of any spacer may be the same asa portion of a sequence of another spacer. In some embodiments, at leasta portion of some of the spacers is the same.

In some embodiments, any epitope tag construct comprises at least onespacer comprising the sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, or SEQ ID NO: 13, SEQ ID NO: 14 (or at least a portion of theamino acid sequence constituting the spacer compiles an amino acidsequence including a sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, or SEQ ID NO: 14). In other embodiments, anyepitope tag construct comprises at least one spacer comprising thesequence of one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, or SEQ ID NO: 14 (or at least a portion of the amino acidsequence constituting the spacer comprises an amino acid sequenceincluding a sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, or SEQ ID NO: 14); and at least a second spacer comprisingthe sequence of another one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, or SEQ ID NO: 14 (or at least a portion of the aminoacid sequence constituting the spacer comprises an amino acid sequenceincluding a sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, or SEQ ID NO: 14.

In some embodiments, the molecular weight of an epitope tag constructranges from between about 5 g/mol to about 35 g/mol. In otherembodiments, the molecular weight of an epitope tag construct rangesfrom between about 5 g/mol to about 30 g/mol. In other embodiments, themolecular weight of an epitope tag construct ranges from between about10 g/mol to about 30 g/mol. In other embodiments, the molecular weightof an epitope tag construct ranges from between about 5 g/mol to about25 g/mol. In other embodiments, the molecular weight of an epitope tagconstruct ranges from between about 5 g/mol to about 20 g/mol. In otherembodiments, the molecular weight of an epitope tag construct rangesfrom between about 75 g/mol to about 15 g/mol. In other embodiments, themolecular weight of an epitope tag construct ranges does not exceed 20g/mol. In other embodiments, the molecular weight of an epitope tagconstruct ranges does not exceed 15 g/mol.

In some embodiments, a distance between successive -[Epitope Tags]- ofthe at least one epitope tag construct ranges from between about 8 nm toabout 18 nm. In some embodiments, a distance between successive-[Epitope Tags]- of the at least one epitope tag construct is less than18 nm. In some embodiments, a distance between successive -[EpitopeTags]- of the at least one epitope tag construct is less than 16 nm. Insome embodiments, a distance between successive -[Epitope Tags]- of theat least one epitope tag construct is less than 14 nm. In someembodiments, a distance between successive -[Epitope Tags]- of the atleast one epitope tag construct is less than 12 nm. In some embodiments,a distance between successive -[Epitope Tags]- of the at least oneepitope tag construct is less than 11 nm. In some embodiments, adistance between successive -[Epitope Tags]- of the at least one epitopetag construct is less than 10 nm. In some embodiments, the -[Spacer]- issized to facilitate bivalent binding of anti-tag antibodies betweenadjacent Epitope Tags (see FIG. 35A). As such, in some embodiments, anytwo adjacent Epitope Tags are spaced a distance apart from one anotherwhich approximates the distance between antigen binding Sites of ananti-tag antibody. In some embodiments, a first antigen binding sitebinds to a first Epitope Tag of the at least one epitope tag constructand wherein a second antigen binding site binds to a second, adjacentEpitope Tag of the at least one epitope tag construct.

While the spacing may facilitate the bivalent binding of antibodies, theskilled artisan will appreciate that it is also possible for antibodies,e.g. anti-tag antibodies, to “skip” an epitope tag and bind anon-adjacent epitope tag (see FIG. 35B). For example, the epitope tagantibody may comprise first, second, and third epitope tags, where thefirst and second epitope tags are adjacent to one another (but, ofcourse, separated by a spacer). In this example, a first antigen bindingsite of an anti-tag antibody may bind to the first epitope tag and asecond antigen binding site of the anti-tag antibody may bind to thethird epitope tag. The skilled artisan will also appreciate that thecombined flexibility and the length of the spacers may form a spatialarrangement of contiguous or non-contiguous epitopes that could beaccessed by both arms of an anti-tag antibody with elbow angles betweenabout 120 degrees and about 220 degrees. In some embodiments, the elbowangles range from about 120 degrees to about 200 degrees. In someembodiments, the elbow angles range from about 140 degrees to about 200degrees.

In some embodiments, the combined molecular weight of all epitope tagconstructs of any epitope-tagged antibody is between about 5 g/mol toabout 80 g/mol. In other embodiments, the combined molecular weight ofall epitope tag constructs of any epitope-tagged antibody is betweenabout 5 g/mol to about 50 g/mol. In yet other embodiments, the combinedmolecular weight of all epitope tag constructs of any epitope-taggedantibody is between about 10 g/mol to about 40 g/mol. In furtherembodiments, the combined molecular weight of all epitope tag constructsof any epitope-tagged antibody is between about 15 g/mol to about 30g/mol.

In yet further embodiments, the combined molecular weight of all epitopetag constructs of any epitope-tagged antibody is less than 40% of themolecular weight of the native antibody (i.e. the molecular weight of acorresponding unmodified antibody having the same specificity and/orfunctional characteristics). In yet further embodiments, the combinedmolecular weight of all epitope tag constructs of any epitope-taggedantibody is less than 30% of the molecular weight of the nativeantibody. In yet further embodiments, the combined molecular weight ofall epitope tag constructs of any epitope-tagged antibody is less than25% of the molecular weight of the native antibody.

Engineering of Epitope Tag Constructs and Epitope-Tagged Antibodies

The epitope-tagged antibodies of the present disclosure may be generatedaccording to methods known to those of ordinary skill in the art. Ingeneral, an “epitope gene” comprising tandem repeat epitope tagsseparated with spacers is synthesized and the sequence verified. In someembodiments, the epitope gene comprises a nucleic acid sequence of anyof SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ IDNO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31.In other embodiments, the epitope gene comprises a nucleic acid sequencehaving at least 80% identify to any of SEQ ID NO: 15, SEQ ID NO: 17, SEQID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27,SEQ ID NO: 29, or SEQ ID NO: 31. In yet other embodiments, the epitopegene comprises a nucleic acid sequence having at least 90% identify toany of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO:31. In further embodiments, the epitope gene comprises a nucleic acidsequence having at least 95% identify to any of SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31. In yet further embodiments,the epitope gene comprises a nucleic acid sequence having about 97%identify to any of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 orSEQ ID NO: 31.

Following epitope gene synthesis, the epitope gene is cloned into anappropriate vector (e.g. pUC57-Kan) using midi-scale DNA preparation(e.g. to preserve the sequence and to propagate the gene by means of thevector). The epitope gene is then cloned at the C-terminal end of aheavy chain constant region or the C-terminal end of a light chainconstant region of the antibody of interest by means of a restrictionsite at the C-terminal end (e.g. 5′Not1 and 3′Sfil) and inserted intothe multiple cloning site (MCS) of an antibody expression vector. Tomake the final heavy constant chain region—epitope tag construct orfinal light constant chain region—epitope tag construct, maxi-scale DNApreparation is used. The DNA constructs (heavy constant chainregion—epitope tag construct or final light constant chainregion—epitope tag construct) are then transiently transfected intoHEK293 cells via route antibody expression and production processes, asknown to those of ordinary skill in the art.

Alternatively, an antibody expression vector is selected from one thatis currently used in antibody expression and production, i.e. anantibody expression vector used to produce a native, unmodifiedantibody. After the epitope gene is synthesized and inserted into avector, e.g. pUC57, the epitope gene is cloned into the antibodyexpression vector downstream of the antibody gene (e.g. via Not1 andSfil site) with the correct reading frame. In this manner, a library ofepitope vectors may be built using an existing antibody expressionvector. Next the IgG H and K chain gene sequence of the antibody ofinterest are extracted from the antibody expression vector via therestriction sites (e.g. Nhe1 and Not1) and inserted into the epitopevector via the same restriction site (e.g. Nhe1 and Not1). The IgG H orK chain sequence is located upstream of the tag sequence with thecorrect reading frame. In the culture system, the epitope vectorexpresses the IgG H or K chain and the tag at the C-terminal end of IgG.

Examples of Specific Epitope Tag Constructs and Epitope-TaggedAntibodies

Provided herein are specific examples of epitope tag constructs whichmay be engineered to be incorporated at a terminal end of a heavy and/orlight chain constant region of an antibody. The skilled artisan willappreciate that epitope constructs having more or less epitope tags maybe produced by altering the epitope tag gene according to the proceduresdescribed above and according to other methods known to those of skillin the art. As such, the examples which follow are non-limitingexamples.

Epitope-Tagged Antibody Comprising an Epitope Construct Having Four VSVEpitope Tags

In some embodiments is an epitope-tagged antibody that expresses the VSVepitope tag. In some embodiments, an epitope-tagged antibody comprisesat least one epitope tag construct, the at least one epitope tagconstruct including four VSV epitope tags. In some embodiments, theepitope tag construct includes four epitope tags having the amino acidsequence of SEQ ID NO: 1. In some embodiments, the epitope tag constructhas at least one spacer which includes at least one of the amino acidsequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,or SEQ ID NO: 14. In some embodiments, the epitope-tagged antibodycomprises an amino acid sequence of SEQ ID NO: 16. In some embodiments,the epitope-tagged antibody comprises an amino acid sequence of SEQ IDNO: 16 at a C-terminal end of a heavy chain constant region. In othe6 ata C-terminal end of a light chain constant region. In yet otherembodiments, the epitope-tagged antibody comprises an amino acidsequence of SEQ ID NO: 16 at both a C-terminal end of a heavy chainconstant region and a C-terminal end of a light chain constant region.In some embodiments, the epitope-tagged antibody is specific to at leastone of CD3, CD8, CD20, CD68, HER2, FoxP3, PDL1, and EGFR2. In someembodiments, this particular epitope gene may be modified to have, forexample, five epitope tags.

Epitope-Tagged Antibody Comprising an Epitope Construct Having Four AU5Epitope Tags

In some embodiments is an epitope-tagged antibody that express the AU5epitope tag. In some embodiments, an epitope-tagged antibody comprisesat least one epitope tag construct, the at least one epitope tagconstruct including four AU5 epitope tags. In some embodiments, theepitope tag construct includes four epitope tags having the amino acidsequence of SEQ ID NO: 2. In some embodiments, the epitope tag constructhas at least one spacer which includes at least one of the amino acidsequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,of SEQ ID NO: 14. In some embodiments, the epitope-tagged antibodycomprises an amino acid sequence of SEQ ID NO: 18. In some embodiments,the epitope-tagged antibody comprises an amino acid sequence of SEQ IDNO: 18 at a C-terminal end of a heavy chain constant region. In otherembodiments, the epitope-tagged antibody comprises an amino acidsequence of SEQ ID NO: 18 at a C-terminal end of a light chain constantregion. In yet other embodiments, the epitope-tagged antibody comprisesan amino acid sequence of SEQ ID NO: 18 at both a C-terminal end of aheavy chain constant region and a C-terminal end of a light chainconstant region. In some embodiments, the epitope-tagged antibody isspecific to at least one of CD3, CD8, CD20, CD68, HER2, FoxP3, PDL1, andEGFR2. In some embodiments, this particular epitope gene may be modifiedto have, for example, five epitope tags.

Epitope-Tagged Antibody Comprising an Epitope Construct Having Four EEpitope Tags

In some embodiments is an epitope-tagged antibody that expresses the Eepitope tag. In some embodiments, an epitope-tagged antibody comprisesat least one epitope tag construct, the at least one epitope tagconstruct including four E epitope tags. In some embodiments, theepitope tag construct includes four epitope tags having the amino acidsequence of SEQ ID NO: 3. In some embodiments, the epitope tag constructhas at least one spacer which includes at least one of the amino acidsequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,or SEQ ID NO: 14. In some embodiments, the epitope-tagged antibodycomprises an amino acid sequence of SEQ ID NO: 20. In some embodiments,the epitope-tagged antibody comprises an amino acid sequence of SEQ IDNO: 20 at a C-terminal end of a heavy chain constant region. In otherembodiments, the epitope-tagged antibody comprises an amino acidsequence of SEQ ID NO: 20 at a C-terminal end of a light chain constantregion. In yet other embodiments, the epitope-tagged antibody comprisesan amino acid sequence of SEQ ID NO: 20 at both a C-terminal end of aheavy chain constant region and a C-terminal end of a light chainconstant region. In some embodiments, the epitope-tagged antibody isspecific to at least one of CD3, CD8, CD20, CD68, HER2, FoxP3, PDL1, andEGFR2. In some embodiments, this particular epitope gene may be modifiedto have, for example, five epitope tags.

Epitope-Tagged Antibody Comprising an Epitope Construct Having Five V5Epitope Tags

In some embodiments is an epitope-tagged antibody that expresses the V5epitope tag. In some embodiments, an epitope-tagged antibody comprisesat least one epitope tag construct, the at least one epitope tagconstruct including five V5 epitope tags. In some embodiments, theepitope tag construct includes five epitope tags having the amino acidsequence of SEQ ID NO: 4. In some embodiments, the epitope tag constructhas at least one spacer which includes at least one of the amino acidsequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,or SEQ ID NO: 14. In some embodiments, the epitope-tagged antibodycomprises an amino acid sequence of SEQ ID NO: 22. In some embodiments,the epitope-tagged antibody comprises an amino acid sequence of SEQ IDNO: 22 at a C-terminal end of a heavy chain constant region. In otherembodiments, the epitope-tagged antibody comprises an amino acidsequence of SEQ ID NO: 22 at a C-terminal end of a light chain constantregion. In yet other embodiments, the epitope-tagged antibody comprisesan amino acid sequence of SEQ ID NO: 22 at both a C-terminal end of aheavy chain constant region and a C-terminal end of a light chainconstant region. In some embodiments, the epitope-tagged antibody isspecific to at least one of CD3, CD8, CD20, CD68, HER2, FoxP3, PDL1, andEGFR2. In some embodiments, this particular epitope gene may be modifiedto have, for example, four epitope tags.

Epitope-Tagged Antibody Comprising an Epitope Construct Having Four HAEpitope Tags

In some embodiments is an epitope-tagged antibody that expresses HAepitope tags. In some embodiments, an epitope-tagged antibody comprisesat least one epitope tag construct, the at least one epitope tagconstruct including four HA epitope tags. In some embodiments, theepitope tag construct includes four epitope tags having the amino acidsequence of SEQ ID NO: 5. In some embodiments, the epitope tag constructhas at least one spacer which includes at least one of the amino acidsequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,or SEQ ID NO: 14. In some embodiments, the epitope-tagged antibodycomprises an amino acid sequence of SEQ ID NO: 24. In some embodiments,the epitope-tagged antibody comprises an amino acid sequence of SEQ IDNO: 24 at a C-terminal end of a heavy chain constant region. In otherembodiments, the epitope-tagged antibody comprises an amino acidsequence of SEQ ID NO: 24 at a C-terminal end of a light chain constantregion. In yet other embodiments, the epitope-tagged antibody comprisesan amino acid sequence of SEQ ID NO: 24 at both a C-terminal end of aheavy chain constant region and a C-terminal end of a light chainconstant region. In some embodiments, the epitope-tagged antibody isspecific to at least one of CD3, CD8, CD20, CD68, HER2, FoxP3, PDL1, andEGFR2. In some embodiments, this particular epitope gene may be modifiedto have, for example, five epitope tags.

Epitope-Tagged Antibody Comprising an Epitope Construct Having Four E2Epitope Tags

In some embodiment is an epitope-tagged antibody that expresses the E2epitope tag. In some embodiments, an epitope-tagged antibody comprisesat least one epitope tag construct, the at least one epitope tagconstruct including four E2 epitope tags. In some embodiments, theepitope tag construct includes four epitope tags having the amino acidsequence of SEQ ID NO: 6. In some embodiments, the epitope tag constructhas at least one spacer which includes at least one of the amino acidsequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID 13, orSEQ ID NO: 14. In some embodiments, the epitope-tagged antibodycomprises an amino acid sequence of SEQ ID NO: 26. In some embodiments,the epitope-tagged antibody comprises an amino acid sequence of SEQ IDNO: 26 at a C-terminal end of a heavy chain constant region. In otherembodiments, the epitope-tagged antibody comprises an amino acidsequence of SEQ ID NO: 26 at a C-terminal end of a light chain constantregion. In yet other embodiments, the epitope-tagged antibody comprisesan amino acid sequence of SEQ ID NO: 26 at both a C-terminal end of aheavy chain constant region and a C-terminal end of a light chainconstant region. In some embodiments, the epitope-tagged antibody isspecific to at least one of CD3, CD8, CD20, CD68, HER2, FoxP3, PDL1, andEGFR2. In some embodiments, this particular epitope gene may be modifiedto have, for example, five epitope tags.

Epitope-Tagged Antibody Comprising an Epitope Construct Having Four KT3Epitope Tags

In some embodiment is an epitope-tagged antibody that expresses the KT3epitope tag. In some embodiments, an epitope-tagged antibody comprisesat least one epitope tag construct, the at least one epitope tagconstruct including four KT3 epitope tags. In some embodiments, theepitope tag construct includes four epitope tags having the amino acidsequence of SEQ ID NO: 7. In some embodiments, the epitope tag constructhas at least one spacer which includes at least one of the amino acidsequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,or SEQ ID NO: 14. In some embodiments, the epitope-tagged antibodycomprises an amino acid sequence of SEQ ID NO: 28. In some embodiments,the epitope-tagged antibody comprises an amino acid sequence of SEQ IDNO: 28 at a C-terminal end of a heavy chain constant region. In otherembodiments, the epitope-tagged antibody comprises an amino acidsequence of SEQ ID NO: 28 at a C-terminal end of a light chain constantregion. In yet other embodiments, the epitope-tagged antibody comprisesan amino acid sequence of SEQ ID NO: 28 at both a C-terminal end of aheavy chain constant region and a C-terminal end of a light chainconstant region. In some embodiments, the epitope-tagged antibody isspecific to at least one of CD3, CD8, CD20, CD68, HER2, FoxP3, PDL1, andEGFR2. In some embodiments, this particular epitope gene may be modifiedto have, for example, five epitope tags.

Epitope-Tagged Antibody Comprising an Epitope Construct Having Four AU1Epitope Tags

In some embodiment is an epitope-tagged antibody that expresses the AU1epitope tag. In some embodiments, an epitope-tagged antibody comprisesat least one epitope tag construct, the at least one epitope tagconstruct including four AU1 epitope tags. In some embodiments, theepitope tag construct includes four epitope tags having the amino acidsequence of SEQ ID NO: 8. In some embodiments, the epitope tag constructhas at least one spacer which includes at least one of the amino acidsequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,or SEQ ID NO: 14. In some embodiments, the epitope-tagged antibodycomprises an amino acid sequence of SEQ ID NO: 32. In some embodiments,the epitope-tagged antibody comprises an amino acid sequence of SEQ IDNO: 32 at a C-terminal end of a heavy chain constant region. In otherembodiments, the epitope-tagged antibody comprises an amino acidsequence of SEQ ID NO: 32 at a C-terminal end of a light chain constantregion. In yet other embodiments, the epitope-tagged antibody comprisesan amino acid sequence of SEQ ID NO: 32 at both a C-terminal end of aheavy chain constant region and a C-terminal end of a light chainconstant region. In some embodiments, the epitope-tagged antibody isspecific to at least one of CD3, CD8, CD20, CD68, HER2, FoxP3, PDL1, andEGFR2. In some embodiments, this particular epitope gene may be modifiedto have, for example, five epitope tags.

Epitope-Tagged Antibody Comprising an Epitope Construct Having FourOLLAS Epitope Tags

In some embodiment is an epitope-tagged antibody that expresses theOLLAS epitope tag. In some embodiments, an epitope-tagged antibodycomprises at least one epitope tag construct, the at least one epitopetag construct including four OLLAS epitope tags. In some embodiments,the epitope tag construct includes four epitope tags having the aminoacid sequence of SEQ ID NO: 9. In some embodiments, the epitope tagconstruct has at least one spacer which includes at least one of theamino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, or SEQ ID NO: 14. In some embodiments, the epitope-taggedantibody comprises an amino acid sequence of SEQ ID NO: 30. In someembodiments, the epitope-tagged antibody comprises an amino acidsequence of SEQ ID NO: 30 at a C-terminal end of a heavy chain constantregion. In other embodiments, the epitope-tagged antibody comprises anamino acid sequence of SEQ ID NO: 30 at a C-terminal end of a lightchain constant region. In yet other embodiments, the epitope-taggedantibody comprises an amino acid sequence of SEQ ID NO: 30 at both aC-terminal end of a heavy chain constant region and a C-terminal end ofa light chain constant region. In some embodiments, the epitope-taggedantibody is specific to at least one of CD3, CD8, CD20, CD68, HER2,FoxP3, PDL1, and EGFR2. In some embodiments, this particular epitopegene may be modified to have, for example, five epitope tags.

Characterization

The epitope-tagged antibodies disclosed herein, as well as their native,unmodified counterparts, can be detected with anti-species secondaryantibodies in ELISA and IHC. In an ELISA study, an epitope peptide wascoated on the plate, the epitope-tagged primary antibody was applied,followed with goat anti-rabbit secondary antibodies conjugated tohorseradish peroxidase, and 3,3′-Diaminobenzidine (DAB) was used as achromogenic substrate for the enzyme. In IHC, the epitope-tagged primaryantibody was applied to human tonsil tissue, followed with gnatanti-rabbit secondary antibodies conjugated to horseradish peroxidase,and 3,3′-Diaminobenzidine (DAB) was used as a chromogenic substrate forthe enzyme. The primary antibodies (i.e. epitope-tagged antibodies andthe corresponding native, unmodified antibodies) both were tested at 1μg/mL concentrations (see FIGS. 16A through 16F and FIGS. 17A through17F). Only the anti-CD3 epitope-tagged antibody (see FIG. 17D) performedless than ideally and, without wishing to be bound by any particulartheory, it is speculated that the presence of four epitope tags at theC-terminal end of the heavy chain constant region and an additional fourepitope tags at the C-terminal end of the light chain constant regionmay have introduced a steric hindrance which prevented the antibody fromfunctioning ideally, although it still did function in staining assays.

Moreover, as shown in FIG. 18, Applicants have demonstrated that epitopetagging of antibodies has no negative impact on the yield of antibodyproduction as compared with the yield of antibody production ofcounterpart native antibodies. Additionally, the epitope-taggedantibodies were shown, in an accelerated stability study, to be stablefor 24-months (equivalent to 4° C. storage in the accelerated stabilitystudy shown in FIG. 19). The Arrhenius model was followed to predictreal-time stability of tested antibodies (1 ug/ml concentration indiluent 90103) at intended storage conditions (i.e. 4° C.) based on datacollected at elevated temperatures (i.e. 37° C. and 45° C.) for shorterperiods (e.g. 5 to 10 days).

Applicants have also conducted tests utilizing surface plasmon resonanceto determine binding kinetics between anti-tag antibodies and the tandemtags present on the disclosed epitope-tagged antibodies. As noted inExample 12, Applicants have discovered that the kinetics are aviditycatalyzed between the anti-tag antibodies and the tandem tags on theepitope-tagged antibodies (see FIGS. 31A through 31E). As noted herein,“avidity” refers to the overall stability of a complex between two ormore populations of molecules (e.g. an anti-tag antibody and therespective tag), i.e., the functional combining strength of aninteraction between the two populations of molecules. “Aviditycatalyzed” here refers to the cooperative and synergistic bonding ofanti-tag antibodies to tandem tags, possibly, two or more than two tags,in the epitope-tagged antibodies. For example, binding of an anti-V5antibody to the tandem V5 tags on a recombinant antibody of the presentdisclosure, e.g. a CD68 epitope-tagged antibody, has been found to be atleast 240-fold avidity catalyzed to a single V5 tag (see FIG. 31A).Likewise, binding of an anti-HA antibody to the tandem HA tags on arecombinant antibody of the present disclosure, e.g. a CD20epitope-tagged antibody, has been found to be at least 2000-fold aviditycatalyzed to a single HA tag (see FIG. 31D). Without wishing to be boundby any particular theory, it is believed that such kineticssignificantly improve secondary antibody performance. In addition,Applicants believe that the association rates between the anti-tagantibodies and the tags of the epitope-tagged antibodies are rapid andthat the dissociation rates are either out of the instrument'sspecification or were drifting positively for all tag configurationsstudied (see FIGS. 32A through 32D).

Applicants have also conducted (a) dynamic light scattering (DLS)experiments to determine (i) hydrodynamic radii of the epitope-taggedantibodies in solution, and (ii) temperature induced aggregation; (b)differential scanning calorimetry (DSC) experiments to determine theinfluence of the epitope tags on melting temperatures of IgG domains;and (c) SEC experiments to determine the homogeneity of the samples. Byvirtue of the DSC experiments, Applicants have discovered that therecombinant epitope-tagged antibodies of the present disclosure occur asmonomeric immunoglobulins G (IgG) in solution, consistent with theexpected DSC pattern for such a monomeric antibody (see FIGS. 28Athrough 28E). From this, Applicants concluded that the tags of theepitope-tagged antibodies did not disturb the proper folding of theantibody as a whole. As such, the epitope-tagged antibodies maintainedproper folding consisting with the primary antibodies from which theywere derived, while also adding flexibility to the tandem tags andfurther allowing accessibility to the epitope tags.

DLS is a technique that may be used to determine the size distributionprofile of small particles in suspension, or polymers, e.g.biomolecules, in solution. The DLS experiments conducted by Applicantshave shown that the hydrodynamic radius of the epitope-tagged antibodiessuggests the relatively unstructured and very flexible nature of thetags, which allow adequate accessibility to the anti-tag antibodies.Applicants have shown that at temperatures greater than 70° C., theuntagged antibodies formed substantially large aggregates (i.e. greaterthan 1000 nm) and precipitated. In contrast, the presently disclosedepitope tagged-antibodies formed particles with hydrodynamic radii ofbetween about 7 nm and about 25 nm. These panicles were smaller in thecase of epitope-tagged antibodies having tags on both the heavy andlight chains, as compared with those epitope-tagged antibodies havingtags on only the heavy chain or only the light chain. Notably,Applicants observed no significant differences between the various typesof epitope tags incorporated into the epitope-tagged antibodies,including each of the types of tags disclosed herein (see FIG. 26, andalso FIGS. 29A through 29E).

The DLS and DSC data also indicated that incorporation of the epitopetags within the light chain of the antibody at least partiallydestabilized the Fab/CH2 domain and resulted in a decrease in theT_(agg) value up to 4L. In some instances, the DLS data showed a slightincrease in T_(agg) of about 1 K and DSC measurements, suggesting anincrease of the transition temperature (0.1 K or less) of Peak 1 (seeFIGS. 28A through 28E).

As noted above, Applicants further collected SEC data, including datacorresponding to molecular weight determination by right anglescattering (RALS) (see, for example, FIGS. 27A, 27B, and 27C). This dataalso indicated that all of the tested untagged antibodies and thecorresponding epitope-tagged antibodies appeared in solution asmonomeric IgGs. Applicants found that the untagged antibodies had amolecular weight ranging from about 14 kDa to about 150 kDa. In someembodiments, those epitope-tagged antibodies having only heavy chaintags or only light chain tags had molecular weights ranging from about170 kDa to about 210 kDa. In yet other embodiments, those epitope-taggedantibodies having only heavy chain tags or only light chain tags hadmolecular weights ranging from about 185 kDa to about 205 kDa. In otherembodiments, those epitope-tagged antibodies having heavy chain tags oronly light chain tags had molecular weights of about 190 kDa. In someembodiments, the epitope-tagged antibodies having both heavy and lightchain tags had a molecular weight ranging from between 215 kDa to about235 kDa. In some embodiments, the epitope-tagged antibodies having bothheavy and light chain tags had a molecular weight of between about 220kDa and about 230 kDa. In addition, SEC data, like the DLS data notedabove, suggested that the epitope-tagged antibodies were relativelyunstructured and flexible.

When taken together, the results of the DLS, DSC, and SEC experimentsindicated that the flexible tags of the epitope-tagged antibodiesreduced or prevented the formation of large aggregates, such as whenexposed to thermal stresses. It was also able to be concluded that theinclusion of the epitope tags into the antibodies did not disturb theproper folding of the antibody as its whole.

As such, Applicants have shown that epitope-tagged antibodies are stableand, given that they do not exhibit any significant differences instaining as compared with their unmodified, native counterparts, aresuitable for use in IHC assays and especially multiplex IHC assays. Inaddition, Applicants have demonstrated that incorporating epitope tagsonto or within the heavy chain of an antibody provides an advantage interms of thermal stability and quality as compare with incorporating thetags onto the light chain. As will be shown further herein, theepitope-tagged antibodies are believed to be able to be pooled togetherwithout cross-reactivity observed with other native antibodies.

Detection of Epitope-Tagged Antibodies

In some embodiments, any epitope-tagged antibody may comprise adetectable moiety and thus the epitope-tagged antibody may be directlydetected (e.g. conjugated to a detectable moiety).

In other embodiments, specific reagents are utilized to enable detectionof any epitope-tagged antibody, and hence the targets in a tissuesample. In some embodiments, detection reagents are utilized which arespecific to the particular epitope tag of the epitope-tagged antibody.In some embodiments, the detection reagents comprise a secondaryantibody which is specific for the expressed epitope tags of theepitope-tagged antibody, i.e. the secondary antibody is an anti-epitopeor anti-tag antibody. Each anti-tag antibody is designed to detect aspecific epitope tag, e.g. one of VSV, V5, HA, etc.

For example, an epitope-tagged antibody expressing one or more VSVepitope tags could be detected by an anti-VSV antibody, i.e. an anti-tagantibody that is specific for expressed VSV epitope tags. Likewise, anepitope-tagged antibody expressing one or more AU5 epitope tags could bedetected by an anti-AU5 antibody, i.e. an anti-tag antibody that isspecific for expressed AU5 epitope tags.

In some embodiments, the anti-tag antibody may be conjugated to a“detectable moiety” to effectuate detection of the epitope-taggedantibody. A “detectable moiety” is a molecule or material that canproduce a detectable (such as visually, electronically or otherwise)signal that indicates the presence (i.e. qualitative analysis) and/orconcentration (i.e. quantitative analysis) of the epitope-taggedantibody in a sample. A detectable signal can be generated by any knownor yet to be discovered mechanism including absorption, emission and/orscattering of a photon (including radio frequency, microwave frequency,infrared frequency, visible frequency and ultra-violet frequencyphotons).

In some embodiments, the detectable moiety of the anti-tag antibodyincludes chromogenic, fluorescent, phosphorescent and luminescentmolecules and materials, catalysts such as enzymes) that convert onesubstance into another substance to provide a detectable difference(such as by converting a colorless substance into a colored substance orvice versa, or by producing a precipitate or increasing sampleturbidity), haptens that can be detected through antibody-hapten bindinginteractions using additional detectably labeled antibody conjugates,and paramagnetic and magnetic molecules or materials. Of course, thedetectable moieties can themselves also be detected indirectly, e.g. ifthe detectable moiety is a hapten, then yet another antibody specific tothat detectable moiety may be utilized in the detection of thedetectable moiety, as known to those of ordinary skill in the art.

In some embodiments, the anti-tag antibody includes a detectable moietyselected from the group consisting of DAB; AEC; CN; BCIP/NBT; fast red;fast blue; fuchsin; NBT; ALK GOLD; Cascade Blue acetyl azide;Dapoxylsulfonic acid/carboxylic acid succinimidyl ester; DY-405; AlexaFluor 405 succinimidyl ester; Cascade Yellow succinimidyl ester;pyridyloxazole succinimidyl ester (PyMPO); Pacific Blue succinimidylester; DY-415; 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester;DYQ-425; 6-FAM phosphoramidite; Lucifer Yellow; iodoacetamide; AlexaFluor 430 succinimidyl ester; Dabcyl succinimidyl ester; NBDchloride/fluoride; QSY 35 succinimidyl ester; DY-485XL; Cy2 succinimidylester, DY-490; Oregon Green 488 carboxylic acid succinimidyl ester;Alexa Fluor 488 succinimidyl ester; BODIPY 493/503 C3 succinimidylester; DY-480XL; BODIPY FL C3 succinimidyl ester; BODIPY FL C5succinimidyl ester; BODIPY FL-X succinimidyl ester; DYQ-505; OregonGreen 514 carboxylic acid succinimidyl ester; DY-510XL; DY-481XL;6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein succinimidyl ester(JOE); DY-520XL; DY-521XL; BODIPY R6G C3 succinimidyl ester; erythrosinisothiocyanate; 5-carboxy-2′,4′,5′,7′tetrabromosulfonefluoresceinsuccinimidyl ester; Alexa Fluor 532 succinimidyl ester;6-carboxy-2′,4,4′,5′7′,7′-hexachlorofluorescein succinimidyl ester(HEX); BODIPY 530/550 C3 succinimidyl ester; DY-530; BODIPY TMR-Xsuccinimidyl ester; DY-555; DYQ-1; DY-556; Cy3 succinimidyl ester;DY-547; DY-549; DY-550; Alexa Fluor 555 succinimidyl ester; Alexa Fluor546 succinimidyl ester; DY-548; BODIPY 558/568 C3 succinimidyl ester;Rhodamine red-X succinimidyl ester; QSY 7 succinimidyl ester; BODIPY564/570 C3 succinimidyl ester; BODIPY 576/589 C3 succinimidyl ester;carboxy-X-rhodamine (ROX); succinimidyl ester; Alexa Fluor 568succinimidyl ester; DY-590; BODIPY 581/591 C3 succinimidyl ester;DY-591; BODIPY TR-X succinimidyl ester; Alexa Fluor 594 succinimidylester; DY-594; carboxynaphthofluorescein succinimidyl ester; DY-605;DY-610; Alexa Fluor 610 succinimidyl ester; DY-615; BODIPY 630/650-Xsuccinimidyl ester; erioglaucine; Alexa Fluor 633 succinimidyl ester;Alexa Fluor 635 succinimidyl ester; DY-634; DY-630; DY-631; DY-632;DY-633; DYQ-2; DY-636; BODIPY 650/665-X succinimidyl ester; DY-635; Cy5succinimidyl ester; Alexa Fluor 647 succinimidyl ester; DY-647; DY-648;DY-650; DY-654; DY-652; DY-649; DY-651; DYQ-660; DYQ-661; Alexa Fluor660 succinimidyl ester; Cy5.5 succinimidyl ester; DY-667; DY-675;DY-676; DY-678; Alexa Fluor 680 succinimidyl ester; DY-679; DY-680;DY-682; DY-681; DYQ-3; DYQ-700; Alexa Fluor 700 succinimidyl ester;DV-703; DY-701; DY-704; DY-700; DY-730; DY-731; DY-732; DY-734; DY-750;Cy7 succinimidyl ester; DY-74; DYQ-4; and Cy7.5 succinimidyl ester.

Fluorophores belong to several common chemical classes includingcoumarins, fluoresceins (or fluorescein derivatives and analogs),rhodamines, resorufins, luminophores and cyanines. Additional examplesof fluorescent molecules can be found in Molecular Probes Handbook—AGuide to Fluorescent Probes and Labeling Technologies, Molecular Probes,Eugene, Oreg., TheroFisher Scientific, 11^(th) Edition. In otherembodiments, the fluorophore is selected from xanthene derivatives,cyanine derivatives, squaraine derivatives, naphthalene derivatives,coumarin derivatives, oxadiazole derivatives, anthracene derivatives,pyrene derivatives, oxazine derivatives, acridine derivatives,arylmethine derivatives, and tetrapyrrole derivatives. In otherembodiments, the fluorescent moiety is selected from a CF dye (availablefrom Biotium), DRAQ and CyTRAK probes (available from BioStatus), BODIPY(available from Invitrogen), Alexa Fluor (available from Invitrogen),DyLight Fluor (e.g. DyLight 649) (available from Thermo Scientific,Pierce), Atto and Tracy (available from Sigma Aldrich), FluoProbes(available from Interchim), Abberior Dyes (available from Abberior), DYand MegaStokes Dyes (available from Dyomics), Sulfo Cy dyes (availablefrom Cyandye), HiLyte Fluor (available from AnaSpec), Seta, SeTau andSquare Dyes (available from SETA BioMedicals) Quasar and Cal Fluor dyes(available from Biosearch Technologies), SureLight Dyes (available fromAPC, RPEPerCP, Phycobilisomes) (Columbia Biosciences), and APC, APCXL,RPE, BPE (available from Phyco-Biotech, Greensea, Prozyme, Flogen).

In some embodiments, the epitope-tagged antibody is an anti-biomarkerantibody, and detection of the biomarker is facilitated by contactingthe sample with an anti-tag specific binding agent (such as an anti-tagantibody) adapted to deposit a detectable moiety in close proximity tothe anti-biomarker antibody when bound to the sample. In someembodiments, the anti-tag antibody is directly conjugated to thedetectable moiety (hereafter, “direct method”). In other embodiments,the detectable moiety is indirectly associated with the anti-tagspecific binding agent (hereafter, “indirect method”). In someembodiments, the detection reagents are suitable for an indirect method,wherein the detectable moiety is deposited via an enzymatic reactionlocalized to the biomarker via an anti-biomarker antibody/anti-tagspecific binding agent complex. Suitable enzymes for such reactions arewell-known and include, but are not limited to, oxidoreductases,hydrolases, and peroxidases. Specific enzymes explicitly included arehorseradish peroxidase (HRP), alkaline phosphatase (AP), acidphosphatase, glucose oxidase, β-galactosidase, β-glucuronidase, andβ-lactamase. The enzyme may be directly conjugated to the anti-tagantibody, or may be indirectly associated with the anti-tag antibody viaa labeling conjugate. As used herein, a “labeling conjugate” comprises:

(a) a specific detection reagent; and

(b) an enzyme conjugated to the specific detection reagent, wherein theenzyme is reactive with the chromogenic substrate, signaling conjugate,or enzyme-reactive dye under appropriate reaction conditions to effectin situ generation of the dye and/or deposition of the dye on the tissuesample.

As used herein, the term “specific detection reagent” shall refer to anycomposition of matter that is capable of specifically binding to atarget chemical structure in the context of a cellular sample. As usedherein, the phrase “specific binding,” “specifically binds to,” or“specific for” or other similar iterations refers to measurable andreproducible interactions between a target and a specific detectionreagent, which is determinative of the presence of the target in thepresence of a heterogeneous population of molecules including biologicalmolecules. For example, an antibody that specifically binds to a targetis an antibody that binds this target with greater affinity, avidity,more readily, and/or with greater duration than it binds to othertargets. In one embodiment, the extent of binding of a specificdetection reagent to an unrelated target is less than about 10% of thebinding of the antibody to the target as measured, e.g., by aradioimmunoassay (RIA). In certain embodiments, a biomarker-specificreagent that specifically binds to a target has a dissociation constant(Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In anotherembodiment, specific binding can include, but does not require exclusivebinding. Exemplary specific detection reagents include nucleic acidprobes specific for particular nucleotide sequences; antibodies andantigen binding fragments thereof; and engineered specific bindingcompositions, including ADNECTINs (scaffold based on 10th FN3fibronectin; Bristol-Myers-Squibb Co.), AFFIBODYs (scaffold based on Zdomain of protein A from S. aureus; Affibody AB, Solna, Sweden), AVIMERs(scaffold based on domain A/LDL receptor; Amgen, Thousand Oaks, Calif.),dAbs (scaffold based on VH or VL antibody domain, GlaxoSmithKline PLC,Cambridge, UK), DARPins (scaffold based on Ankyrin repeat proteins;Molecular Partners AG, Zürich, CH), ANTICALINs (scaffold based onlipocalins; Pieris AG, Freising, DE), NANOBODYs (scaffold based on VHH(camelid Ig); Ablynx N/V, Ghent, BE), TRANS-BODYs (scaffold based onTransferrin; Pfizer Inc., New York, N.Y.), SMIPs (Emergent Biosolutions,Inc., Rockville, Md.), and TETRANECTINs (scaffold based on C-type lectindomain (CTLD), tetranectin; Borean Pharma A/S, Aarhus, DK). Descriptionsof such engineered specific binding structures are reviewed by Wurch etal., Development of Novel Protein Scaffolds as Alternatives to WholeAntibodies for Imaging and Therapy: Status on Discovery Research andClinical Validation, Current Pharmaceutical Biotechnology, Vol. 9, pp.502-509 (2008), the content of which is incorporated by reference. Innon-limiting examples, the specific detection reagent of the labelingconjugate may be a secondary detection reagent (such as aspecies-specific secondary antibody bound to an anti-tag antibody, ananti-tag antibody bound to an epitope-tagged anti-tag antibody specificfor the anti-biomarker antibody, an anti-hapten antibody bound to ahapten-conjugated anti-tag antibody, or a biotin-binding protein boundto a biotinylated anti-tag antibody antibody), or other sucharrangements. An enzyme thus localized to the sample-boundanti-biomarker antibody can then be used in a number of schemes todeposit a detectable moiety.

In some cases, the enzyme reacts with a chromogenic compound/substrate.Particular non-limiting examples of chromogenic compounds/substratesinclude 4-nitrophenylphospate (pNPP), fast red, bromochloroindolylphosphate (BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, fast red, APOrange, AP blue, tetramethylbenzidine (TMB),2,2′-azino-di-[3-ethylbenzothiazoline sulphonate] (ABTS), o-dianisidine,4-chloronaphthol (4-CN), nitrophenyl-β-D-galactopyranoside (ONPG),o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-βgalactopyranoside(X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal),p-nitrophenyl-α-D-galactopyranoside (PNP),5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazoluim blue,or tetrazolium violet.

In some embodiments, the enzyme can be used in a metallographicdetection scheme. Metallographic detection methods include using anenzyme such as alkaline phosphatase in combination with a water-solublemetal ion and a redox-inactive substrate of the enzyme. In someembodiments, the substrate is converted to a redox-active agent by theenzyme, and the redox-active agent reduces the metal ion, causing it toform a detectable precipitate. (see, for example, U.S. patentapplication Ser. No. 11/015,646, filed Dec. 20, 2004, PCT PublicationNo. 2005/003777 and U.S. Patent Application Publication No.2004/0265922; each of which is incorporated by reference herein in itsentirety). Metallographic detection methods include using anoxido-reductase enzyme (such as horseradish peroxidase) along with awater soluble metal ions, oxidizing agent and a reducing agent, again tofor form a detectable precipitate. (See, for example, U.S. Pat. No.6,670,113, which is incorporated by reference herein in its entirety).

In some embodiments, the enzymatic action occurs between the enzyme andthe dye itself, wherein the reaction converts the dye from a non-bindingspecies to a species deposited on the sample. For example, reaction ofDAB with a peroxidase (such as horseradish peroxidase) oxidizes the DAB,causing it to precipitate.

In yet other embodiments, the detectable moiety is deposited via asignaling conjugate comprising a latent reactive moiety configured toreact with the enzyme to form a reactive species that can bind to thesample or to other detection components. These reactive species arecapable of reacting with the sample proximal to their generation, i.e.near the enzyme, but rapidly convert to a non-reactive species so thatthe signaling conjugate is not deposited at sites distal from the siteat which the enzyme is deposited. Examples of latent reactive moietiesinclude: quinone methide (QM) analogs, such as those described atWO2015124702A1, and tyramide conjugates, such as those described at,WO2012003476A2, each of which is hereby incorporated by reference hereinin its entirety. In some examples, the latent reactive moiety isdirectly conjugated to a dye, such asN,N′-biscarboxypentyl-5,5′-disulfonato-indo-dicarbocyanine (Cy5),4-(dimethylamino) azobenzene-4′-sulfonamide (DABSYL),tetramethylrhodamine (DISCO Purple), and Rhodamine 110 (Rhodamine). Inother examples, the latent reactive moiety is conjugated to one memberof a specific binding pair, and the dye is linked to the other member ofthe specific binding pair. In other examples, the latent reactive moietyis linked to one member of a specific binding pair, and an enzyme islinked to the other member of the specific binding pair, wherein theenzyme is (a) reactive with a chromogenic substrate to effect generationof the dye, or (b) reactive with a dye to effect deposition of the dye(such as DAB). Examples of specific binding pairs include:

(1) a biotin or a biotin derivative (such as desthiobiotin) linked tothe latent reactive moiety, and a biotin-binding entity (such as avidin,streptavidin, deglycosylated avidin (such as NEUTRAVIDIN), or a biotinbinding protein having a nitrated tyrosine at its biotin binding site(such as CAPTAVIDIN)) linked to a dye or to an enzyme reactive with achromogenic substrate or reactive with a dye (for example, a peroxidaselinked to the biotin-binding protein when the dye is DAB);

(2) epitope tags and associated anti-tag antibodies; and

(2) a hapten linked to the latent reactive moiety, and an anti-haptenantibody linked to a dye or to an enzyme reactive with a chromogenicsubstrate or reactive with a dye (for example, a peroxidase linked tothe biotin-binding protein when the dye is DAB).

Non-limiting examples of anti-tag antibody and detection reagentcombinations are set forth in Table 1:

TABLE 1 A. Anti-tag antibody linked directly to detectable moietyAnti-tag antibody-Dye conjugate B. Anti-tag antibody linked to enzymereacting with detectable moiety Anti-tag antibody-Enzyme conjugate + DABAnti-tag antibody-Enzyme conjugate + Chromogen C. Anti-tag antibodylinked to Enzyme reacting with detectable moiety C1. Signaling conjugateAnti-tag antibody-Enzyme conjugate + QM-Dye conjugate comprisesdetectable moiety Anti-tag antibody-Enzyme conjugate + Tyramide-Dyeconjugate C2. Signaling conjugate Anti-tag antibody-Enzyme conjugate +QM-Enzyme comprises enzyme that reacts conjugate + DAB directly withdetectable Anti-tag antibody-Enzyme conjugate + QM-Enzyme moietyconjugate + Chromogen Anti-tag antibody-Enzyme conjugate +Tyramide-Enzyme conjugate + DAB Anti-tag antibody-Enzyme conjugate +Tyramide-Enzyme conjugate + Chromogen C3. Signaling conjugate Anti-tagantibody-Enzyme conjugate + QM-Enzyme comprises enzyme that reactsconjugate + QM-Dye conjugate with second signaling Anti-tagantibody-Enzyme conjugate + QM-Enzyme conjugate comprising conjugate +Tyramide-Dye conjugate detectable moiety Anti-tag antibody-Enzymeconjugate + Tyramide-Enzyme conjugate + QM-Dye conjugate Anti-tagantibody-Enzyme conjugate + Tyramide-Enzyme conjugate + Tyramide-Dyeconjugate C4. Signaling conjugate Anti-tag antibody-Enzyme conjugate +Tyramide-(biotin or comprises member of a epitope tag or hapten)conjugate + Dye-(avidin or anti-tag specific binding pair and antibodyor anti-hapten antibody) conjugate other member of binding pair Anti-tagantibody-Enzyme conjugate + QM-(biotin or is linked to detectable moietyepitope tag or hapten) conjugate + Dye-(avidin or anti-tag antibody oranti-hapten antibody) conjugate C5. Signaling conjugate Anti-tagantibody-Enzyme conjugate + QM-(biotin or comprises member of a epitopetag or hapten) conjugate + Enzyme-(avidin or specific binding pair andanti-tag antibody or anti-hapten antibody) conjugate + other member ofbinding pair DAB is linked to enzyme reactive Anti-tag antibody-Enzymeconjugate + QM-(biotin or with detectable moiety epitope tag or hapten)conjugate + Enzyme-(avidin or anti-tag antibody or anti-hapten antibody)conjugate + Chromogen Anti-tag antibody-Enzyme conjugate +Tyramide-(biotin or epitope tag or hapten) conjugate + Enzyme-(avidin oranti-tag antibody or anti-hapten antibody) conjugate + DAB Anti-tagantibody-Enzyme conjugate + Tyramide-(biotin or epitope tag or hapten)conjugate + Enzyme-(avidin or anti-tag antibody or anti-hapten antibody)conjugate + Chromogen C6. Signaling conjugate Anti-tag antibody-Enzymeconjugate + QM-(biotin or comprises member of a epitope tag or hapten)conjugate + Enzyme-(avidin or specific binding pair and anti-tagantibody or anti-hapten antibody) conjugate + other member of bindingpair Tyramide-Dye conjugate is linked to enzyme reactive Anti-tagantibody-Enzyme conjugate + QM-(biotin or with second detectable epitopetag or hapten) conjugate + Enzyme-(avidin or moiety linked to adetectable anti-tag antibody or anti-hapten antibody) conjugate + moietyQM-Dye conjugate Anti-tag antibody-Enzyme conjugate + Tyramide-(biotinor epitope tag or hapten) conjugate + Enzyme-(avidin or anti-tagantibody or anti-hapten antibody) conjugate + Tyramide-Dye conjugateAnti-tag antibody-Enzyme conjugate + Tyramide-(biotin or epitope tag orhapten) conjugate + Enzyme-(avidin or anti-tag antibody or anti-haptenantibody) conjugate + QM-Dye conjugate D. Anti-tag antibody linked tomember of specific binding pair D1. Dye linked to other Anti-tagantibody-(biotin or epitope tag or hapten) member of specific bindingconjugate + Dye-(avidin or anti-tag antibody or pair anti-haptenantibody) conjugate D2. Enzyme linked to other Anti-tag antibody-(biotinor epitope tag or hapten) member of specific binding conjugate +Enzyme-(avidin or anti-tag antibody or pair, wherein the enzyme isanti-hapten antibody) conjugate + DAB reactive with detectable Anti-tagantibody-(biotin or epitope tag or hapten) moiety conjugate +Enzyme-(avidin or anti-tag antibody or anti-hapten antibody) conjugate +Chromogen Anti-tag antibody-(biotin or epitope tag or hapten)conjugate + Enzyme-(avidin or anti-tag antibody or anti-hapten antibody)conjugate + QM-Dye conjugate Anti-tag antibody-(biotin or epitope tag orhapten) conjugate + Enzyme-(avidin or anti-tag antibody or anti-haptenantibody) conjugate + Tyramide-Dye conjugate E. Secondary detectionreagent linked directly to detectable moiety Anti-tag antibody + 2°specific detection reagent-Dye conjugate F. Secondary detection reagentlinked to Enzyme reacting with detectable moiety Anti-tag antibody + 2°specific detection reagent-Enzyme conjugate + DAB Anti-tag antibody + 2°specific detection reagent-Enzyme conjugate + Chromogen G. Secondarydetection reagent linked to Enzyme reacting with detectable moiety G1.Signaling conjugate Anti-tag antibody + 2° specific detectionreagent-Enzyme compises detectable moiety conjugate + QM-Dye conjugateAnti-tag antibody + 2° specific detection reagent-Enzyme conjugate +Tyramide-Dye conjugate G2. Signaling conjugate Anti-tag antibody +2°specific detection reagent-Enzyme comprises enzyme that reactsconjugate + QM-Enzyme conjugate + DAB directly with detectable Anti-tagantibody + 2° specific detection reagent-Enzyme moiety conjugate +QM-Enzyme conjugate + Chromogen Anti-tag antibody + 2° specificdetection reagent-Enzyme conjugate + Tyramide-Enzyme conjugate + DABAnti-tag antibody + 2° specific detection reagent-Enzyme conjugate +Tyramide-Enzyme conjugate + Chromogen G3. Signaling conjugate Anti-tagantibody + 2° specific detection reagent-Enzyme comprises enzyme thatreacts conjugate + QM-Enzyme conjugate + QM-Dye with second signalingconjugate conjugate comprising Anti-tag antibody + 2° specific detectionreagent-Enzyme detectable moiety conjugate + QM-Enzyme conjugate +Tyramide-Dye conjugate Anti-tag antibody + 2° specific detectionreagent-Enzyme conjugate + Tyramide-Enzyme conjugate + QM-Dye conjugateAnti-tag antibody + 2° specific detection reagent-Enzyme conjugate +Tyramide-Enzyme conjugate + Tyramide-Dye conjugate G4. Signalingconjugate Anti-tag antibody + 2° specific detection reagent-Enzymecomprises member of a conjugate + Tyramide-(biotin or epitope tag orspecific binding pair and hapten) conjugate + Dye-(avidin or anti-tagantibody other member of binding pair or anti-hapten antibody) conjugateis linked to detectable moiety Anti-tag antibody + 2° specific detectionreagent-Enzyme conjugate + QM-(biotin or epitope tag or hapten)conjugate + Dye-(avidin or anti-tag antibody or anti-hapten antibody)conjugate G5. Signaling conjugate Anti-tag antibody + 2° specificdetection reagent-Enzyme comprise member of a conjugate + QM-(biotin orepitope tag or hapten) specific binding pair and conjugate +Enzyme-(avidin or anti-tag antibody or other member of binding pairanti-hapten antibody) conjugate + DAB is linked to enzyme reactiveAnti-tag antibody + 2° specific detection reagent-Enzyme with detectablemoiety conjugate + QM-(biotin or epitope tag or hapten) conjugate +Enzyme-(avidin or anti-tag antibody or anti-hapten antibody) conjugate +Chromogen Anti-tag antibody + 2° specific detection reagent-Enzymeconjugate + Tyramide-(biotin or epitope tag or hapten) conjugate +Enzyme-(avidin or anti-tag antibody or anti-hapten antibody) conjugate +DAB Anti-tag antibody + 2° specific detection reagent-Enzyme conjugate +Tyramide-(biotin or epitope tag or hapten) conjugate + Enzyme-(avidin oranti-tag antibody or anti-hapten antibody) conjugate + Chromogen G6.Signaling conjugate Anti-tag antibody + 2° specific detectionreagent-Enzyme comprises member of a conjugate + QM-(biotin or epitopetag or hapten) specific binding pair and conjugate + Enzyme-(avidin oranti-tag antibody or other member of binding pair anti-hapten antibody)conjugate + Tyramide-Dye is linked to enzyme reactive conjugate withsecond detectable Anti-tag antibody + 2° specific detectionreagent-Enzyme moiety linked to a detectable conjugate + QM-(biotin orepitope tag or hapten) moiety conjugate + Enzyme-(avidin or anti-tagantibody or anti-hapten antibody) conjugate + QM-Dye conjugate Anti-tagantibody + 2° specific detection reagent-Enzyme conjugate +Tyramide-(biotin or epitope tag or hapten) conjugate + Enzyme-(avidin oranti-tag antibody or anti-hapten antibody) conjugate + Tyramide-Dyeconjugate Anti-tag antibody + 2° specific detection reagent-Enzymeconjugate + Tyramide-(biotin or epitope tag or hapten) conjugate +Enzyme-(avidin or anti-tag antibody or anti-hapten antibody) conjugate +QM-Dye conjugate H. Secondary detection reagent linked to member ofspecific binding pair H1. Dye linked to other Anti-tag antibody + 2°specific detection reagent-(biotin or member of specific binding epitopetag or hapten) conjugate + Dye-(avidin or pair anti-tag antibody oranti-hapten antibody) conjugate H2. Enzyme linked to Anti-tag antibody +2° specific detection reagent-(biotin or other member of specificepitope tag or hapten) conjugate + Enzyme-(avidin or binding pair,wherein the anti-tag antibody or anti-hapten antibody) conjugate +enzyme is reactive with DAB detectable moiety Anti-tag antibody + 2°specific detection reagent-(biotin or epitope tag or hapten) conjugate +Enzyme-(avidin or anti-tag antibody or anti-hapten antibody) conjugate +Chromogen Anti-tag antibody + 2° specific detection reagent-(biotin orepitope tag or hapten) conjugate + Enzyme-(avidin or anti-tag antibodyor anti-hapten antibody) conjugate + QM-Dye conjugate Anti-tagantibody + 2° specific detection reagent-(biotin or epitope tag orhapten) conjugate + Enzyme-(avidin or anti-tag antibody or anti-haptenantibody) conjugate + Tyramide-Dye conjugate

As used in Table 1, a “2° specific detection reagent” shall refer to anyentity capable of specific binding to the anti-tag antibody. Thus, forexample, the 2° specific detection reagent may be an anti-speciesantibody that binds specifically to the species of immunoglobulin fromwhich the anti-tag antibody is derived, and anti-hapten antibodyimmunoreactive with a hapten conjugated to the anti-tag antibody, or(where the anti-tag antibody is an epitope-tagged antibody) a secondanti-tag antibody reactive with an epitope-tag of the anti-tag antibody.Furthermore, in each example of Table 1, the anti-tag antibody may besubstituted with another specific detection agent capable of specificbinding to the tag, such as an ADNECTIN, AFFIBODY, AVIMER, dAb, DARPin,ANTICALIN, NANOBODY, TRANS-BODY, SMIP, or a TETRANECTIN.

Detection Kits Comprising Epitope-Tagged Antibodies and DetectionReagents for Detecting Epitope-Tagged Antibodies

In some embodiments, the epitope-tagged antibodies of the presentdisclosure may be utilized as part of a “detection kit.” In general, anydetection kit may include one or more epitope-tagged antibodies anddetection reagents for detecting the one or more epitope-taggedantibodies.

The detection kits may include a first composition comprising anepitope-tagged antibody and a second composition comprising detectionreagents specific to the first composition, such that the epitope-taggedantibody may be detected via the detection kit. In some embodiments, thedetection kit includes a plurality of epitope-tagged antibodies (such ismixed together in a buffer), where the detection kit also includesdetection reagents specific for each of the plurality of epitope-taggedantibodies.

By way of example, a kit may include an epitope-tagged antibody specificfor a first target, the epitope-tagged antibody having a first epitopetag (e.g. VSV), and an epitope-tagged antibody specific for a secondtarget having a second epitope tag (e.g. HA), wherein the first andsecond epitope tags are different. The kit may further comprisedetection reagents specific for each of the different epitope-taggedantibodies. For example, anti-tag antibodies specific for each of theepitope tags of the different epitope-tagged antibodies may be included.In some embodiments, the anti-tag antibodies may be conjugated tofluorescent detectable moieties (e.g. Alexa Fluor IR dyes). In otherembodiments, the anti-tag antibodies may be conjugated to an enzyme, andchromogenic substrates for the enzyme may also be included within anykit.

Of course, any kit may include other agents, including buffers;counterstaining agents; enzyme inactivation compositions;deparrafinization solutions, etc. as needed for manual or automatedtarget detection. The detection kits may also comprise other specificbinding entities (e.g. nucleic acid probes for ISH; unmodified (native)antibodies, and antibody conjugates) and detection reagents to detectthose other specific binding entities. For example, a kit may compriseone or more epitope-tagged antibodies; one or more anti-tag antibodiesfor detecting the one or more epitope-tagged antibodies; at least oneunmodified antibody; and detection reagents for detecting the at leastone unmodified antibody. In some embodiments, instructions are providedfor using the epitope-tagged antibodies, and other components of thekit, for use in an assay, e.g. a MIHC assay.

Methods of Detecting Targets with Epitope-Tagged Antibodies andDetection Reagents

The present disclosure also provides methods of detecting one or moretargets within a tissue using any of the epitope-tagged antibodiesdescribed herein. In some embodiments, an epitope tagged antibody may beused in a simplex assay to detect a particular target within the tissuesample (e.g. CD68, FoxP3, CD20, etc.), where the epitope-tagged antibodyis specific to the target of interest, and where upon application of theepitope-tagged antibody to the tissue sample a target-epitope-taggedantibody complex is formed. Following application of the epitope-taggedantibody, detection reagents (e.g. an anti-tag antibody) are appliedsuch that the target-epitope-tagged antibody complex may be detected. Insome embodiments, the detection reagents comprise an anti-tag antibodyspecific to the particular expressed epitope tag of the epitope-taggedantibody, where the anti-tag antibody comprises a detectable moiety. Thesingle target may then be visualized or otherwise detected.

In some aspects of the present disclosure are provided methods ofmultiplex detection, including automated multiplex detection. FIG. 15Aprovides a flowchart illustrating one method for the multiplex detectionof targets where a tissue sample is contacted simultaneously with aplurality of epitope-tagged antibodies (step 100), where eachepitope-tagged antibody is specific for a particular target, and whereeach epitope-tagged antibody comprises a different epitope tag (adifferent expressed epitope tag).

In some embodiments, the sample may be contacted with two epitope-taggedantibodies, where each epitope-tagged antibody is specific for aparticular target, and where each epitope-tagged antibody comprises adifferent epitope tag. In other embodiments, the sample may be contactedwith three epitope-tagged antibodies, where each epitope-tagged antibodyis specific for a particular target, and where each epitope-taggedantibody comprises a different epitope tag (see, for example, Examples 1and 2). In yet other embodiments, the sample may be contacted with fouror more epitope-tagged antibodies, where each epitope-tagged antibody isspecific for a particular target, and where each epitope-tagged antibodycomprises a different epitope tag (see, for example, Example 7).

The epitope-tagged antibodies may be supplied to the tissue sample as a“pool” or “cocktail” comprising each of the epitope-tagged antibodiesneeded for the particular assay. The pooling of epitope-taggedantibodies is believed to be possible since the epitope-taggedantibodies do not show cross-reactivity to each other, at least not tothe extent where any cross-reactivity would interfere with stainingperformance. Each epitope-tagged antibody will bind to their respectivetargets and form detectable target-epitope-tagged antibody complexes. Insome embodiments, and following application of the epitope-taggedantibodies, a blocking step is performed (see, for examples, Examples 1,2, and 7 herein which illustrate the incorporation of a blockings stepand/or other processing steps).

Following the simultaneous application of the epitope-tagged antibodies(step 100), a plurality of anti-tag antibodies is simultaneously appliedto the tissue sample (step 110), where each anti-tag antibody isspecific to one of the epitope-tagged antibodies initially applied (atstep 100), and where each anti-tag antibody comprises a differentdetectable moiety. In some embodiments, the detectable moiety is afluorophore. The anti-tag antibodies may be supplied to the tissuesample as a pool or cocktail comprising each of the anti-tag antibodiesnecessary for detection of the target-epitope-tagged antibody complexes.Following application of the anti-tag antibodies, in some embodimentsthe tissue sample may be stained with a counterstain. Signals from eachof the detectable moieties may be visualized or otherwise detected (e.g.simultaneously visualized or detected).

As an example of a multiplex assay according to one aspect of thepresent disclosure, a first epitope-tagged antibody comprising a firstepitope tag and specific to a first target (e.g. specific to one ofCD68, FoxP3, CD20, etc.) is introduced to a tissue sample. In someembodiments, the first epitope-tagged antibody forms a detectable firsttarget-epitope-tagged antibody complex. Simultaneously, a secondepitope-tagged antibody comprising a second epitope tag and specific toa second target (e.g. another of CD68, FoxP3, CD20, etc.) is introducedto the sample to form a second target-epitope-tagged antibody complex.Third, fourth, and nth additional epitope-tagged antibodies specific toother targets (forming “n” target-detection probe complexes) and havingdifferent epitope tags may be further introduced simultaneously with thefirst and second epitope-tagged antibody antibodies.

After the epitope-tagged antibodies are deposited, they may be detected,either directly of indirectly depending, of course, on theirconfiguration. In some embodiments, anti-tag antibodies are introducedto enable detection of each of the target-epitope-tagged antibodycomplex. In some embodiments, the anti-tag antibodies are specific tothe different epitope tags of the epitope-tagged antibodies, and wherethe anti-tag antibodies are each conjugated to a detectable moiety. Insome embodiments, the detectable reagents are anti-tag antibodies eachconjugated to a fluorophore. In some embodiments, first, second, and nthanti-tag antibodies are simultaneously introduced, where each of thefirst, second, and nth detection reagents are specific to the differentepitope-tagged antibodies, where each of the anti-tag antibodies areconjugated to a fluorophore. In other embodiments, first, second, andnth anti-tag antibodies are sequentially introduced, where each of thefirst, second, and nth detection reagents are specific to the differentepitope-tagged antibodies, and wherein each of the anti-tag antibodiesare conjugated to an enzyme.

As a further example of a multiplex assay according to the presentdisclosure, a first epitope-tagged antibody specific to a first target(e.g. CD3, FoxP3, PD-L1, or an immune cell marker) is introduced to atissue sample, the first epitope-tagged antibody expressing a firstepitope tag. In some embodiments, the first epitope-tagged antibodyforms a detectable first target-epitope-tagged antibody conjugatecomplex. Either simultaneously or subsequently, a second epitope-taggedantibody specific to a second target (e.g. another of CD3, FoxP3, PD-L1)is introduced to the sample to form a second target-epitope-taggedantibody conjugate complex, the second epitope-tagged antibodyexpressing a second epitope tag. Third, fourth, and nth additionalepitope-tagged antibodies each specific to other targets (forming “n”target-epitope-tagged antibody conjugate complexes) may be furtherintroduced, again either sequentially or simultaneously with the firstand/or second epitope-tagged antibodies, where the third, fourth and nthepitope-tagged antibodies each express yet further different epitopetags. After the epitope-tagged antibodies are deposited, they may bedetected. In some embodiments, additional detection reagents areintroduced to enable the detection of the targets and the additionaldetection reagents include those described herein (e.g. chromogenicdetection reagents). In some embodiments, first, second, and nthdetection reagents are sequentially introduced, where each of the first,second, and nth detection reagents comprise (i) a secondary antibody,namely an anti-tag antibody, specific to each of the epitope tags of theepitope-tagged antibodies, wherein the secondary antibody is conjugatedto an enzyme; and (ii) a chromogenic substrate; wherein each of thefirst, second, and nth chromogenic substrates are different.

In some embodiments, the multiplex detection method comprises the stepsof (i) contacting a biological sample with a first epitope-taggedantibody to form a first target-epitope-tagged antibody conjugatecomplex; (ii) contacting the biological sample with a first labelingconjugate wherein the first labeling conjugate comprises a first enzyme(where the first labeling conjugate is an anti-tag antibody thatspecifically binds to the first epitope-tagged antibody and isconfigured to label the target with an enzyme); (iii) contacting thebiological sample with a first signaling conjugate comprising a firstlatent reactive moiety and a first chromogenic moiety (see, e.g. U.S.patent application Ser. No. 13/849,160, the disclosure of which isincorporated herein by reference for a description of signalingconjugates and their constituent components); (iv) inactivating thefirst enzyme, such as by contacting the sample with a first enzymeinactivation composition to substantially inactivate or completelyinactivate the first enzyme contained in the biological sample.

After the first enzyme is inactivated (optional), the multiplex methodfurther comprises the steps of (v) contacting a biological sample with asecond epitope-tagged antibody to form a second target-epitope-taggedantibody conjugate complex; (vi) contacting the biological sample with asecond labeling conjugate wherein the second labeling conjugatecomprises a second enzyme (where the second labeling conjugate is ananti-tag antibody that specifically binds to the second epitope-taggedantibody and is configured to label the target with an enzyme); (vii)contacting the biological sample with a second signaling conjugatecomprising a second latent reactive moiety and a second chromogenicmoiety; (viii) inactivating the second second enzyme, such as bycontacting the sample with a first enzyme inactivation composition tosubstantially inactivate or completely inactivate the first enzymecontained in the biological sample.

After the second enzyme is inactivated, the method may be repeated suchthat additional epitope-tagged antibodies may be introduced, along withadditional detection reagents, to effectuate detection of other targets.Following introduction of all of the epitope-tagged antibody (and otherdetection probes) and respective detection reagents or kits, the methodfurther comprises the step of counterstaining the sample and/ordetecting signals (manually or via an automated method) from the first,second, and nth chromogenic moieties, wherein each of the first, second,and nth chromogenic moieties are each different. Alternatively, each ofthe epitope-tagged antibodies may be added simultaneously orsequentially, but before any labeling conjugate is added. As anotherexample, three epitope-tagged antibody may be sequentially appliedinitially, prior to introduction of any detection reagents, and theneach of the detection reagents added sequentially.

In the context of a multiplex assay where multiple targets are detectedsequentially, and where the detection employs the use of enzymes, it isdesirable to inactivate any reagent or endogenous enzymes betweensuccessive detection steps. As a result, it is believed that enzymespresent in any one detection step will not interfere with those in alater detection steps. This in turn is believed to improve upon thevisualization and detection of the different detectable moieties used inthe multiplex assay. Any enzyme inactivation composition known in theart may be used for this purpose. In some embodiments, an enzymeinactivation composition is applied to inactivate the reagent orendogenous enzymes after each detection step. Exemplary enzymeinactivation compositions are disclosed in application U.S. 62/159,297,the disclosure of which is incorporated by reference herein in itsentirety.

In some embodiments, a denaturation step presents the enzyme used in afirst set of detection reagents from acting on a second substrate. Insome embodiments, the denaturant is a substance that denatures theenzyme in the first detection reagent set. In some embodiments, thedenaturant is, for example, formamide, an alkyl-substituted amide, ureaor a urea-based denaturant, thiourea, guanidine hydrochloride, orderivatives thereof. Examples of alkyl-substituted amides include, butare not limited to, N-propylformamide, N-butylformamide,N-isobutylformamide, and N,N-dipropylaformamide. In some embodiments,the denaturant is provided in a buffer. For example, formamide may beprovided in a hybridization buffer comprising 20 mM dextran sulfate(50-57% formamide (Ultra Pure formamide stock), 2×SSC (20×SSC stockcontaining 0.3 M citrate and 3M NaCl), 2.5 mM EDTA (0.5M EDTA stock), 5mM Tris, pH 7.4 (1 mM Tris, pH 7.4 stock), 0.05% Brij-35 (10% stockcontaining polyoxyethylene (23) lauryl ether), pH 7.4. In someembodiments, the sample is treated with the denaturant for a period oftime and under conditions sufficient to denature the first target probedetection enzyme, for example alkaline phosphatase. In some embodiments,the sample is heated with the denaturant for about 15 to about 30minutes, preferably about 20 to 24 minutes at about 37° C. In someembodiments, the sample is treated with the denaturant for a period oftime and under conditions sufficient to denature the target enzyme whilepreserving hybridization of the second nucleic acid probe to the target.

For those embodiments employing an anti-tag antibody conjugated to anenzyme, conditions suitable for introducing the signaling conjugates orchromogenic substrates with the biological sample are used, andtypically include providing a reaction buffer or solution that comprisesa peroxide (e.g., hydrogen peroxide), and that has a salt concentrationand pH suitable for allowing or facilitating the enzyme to perform itsdesired function. In general, this step of the method is performed attemperatures ranging from about 35° C. to about 40° C., although theskilled artisan will be able to select appropriate temperature rangesappropriate for the enzymes and signalizing conjugates selected. Forexample, it is believed that these conditions allow the enzyme andperoxide to react and promote radical formation on the latent reactivemoiety of the signaling conjugate. The latent reactive moiety, andtherefore the signaling conjugate as a whole, will deposit covalently onthe biological sample, particularly at one or more tyrosine residuesproximal to the immobilized enzyme conjugate, tyrosine residues of theenzyme portion of the enzyme conjugate, and of tyrosine residues of theantibody portion of the enzyme conjugate. The biological sample is thenilluminated with light and the target may be detected through absorbanceof the light produced by the chromogenic moiety of the signalingconjugate.

Methods of Detection with Epitope-Tagged Antibodies in Conjunction withOther Specific Binding Entities

In some aspects of the present disclosure, epitope-tagged antibodies arein conjugation with other specific binding entities to effect multiplexdetection of targets in a tissue sample. The skilled artisan willappreciate that any of the above-identified methods and procedures maybe adapted accordingly for any assay employing both epitope-taggedantibodies and other specific binding entities.

In some embodiments, the specific binding entities include nucleic acidsfor in situ hybridization, unmodified antibodies for IHC, and/orantibody conjugates for IHC. As used herein, the terms “unmodifiedantibody” or “unmodified antibodies” refer to those antibodies that doesnot comprise an epitope tag or those antibodies which are not conjugatedto any other moiety. In essence, “unmodified antibodies” are nativeantibodies traditionally used in IHC assays, which are specific to aparticular target (e.g. an anti-CD3 antibody) and which may be detected,such as with anti-species secondary antibodies. By way of example, arabbit anti-CD3 antibody may be detected with a goat anti-rabbitantibody.

“Antibody conjugates,” as that term is used herein, refers to thoseantibodies conjugated (either directly or indirectly) to one or morelabels, where the antibody conjugate is specific to a particular targetand where the label is capable of being detected (directly orindirectly), such as with a secondary antibody (an anti-label antibody).For example, an antibody conjugate may be coupled to a hapten such asthrough a polymeric linker and/or spacer, and the antibody conjugate, bymeans of the hapten, may be indirectly detected. As an alternativeexample, an antibody conjugate may be coupled to a fluorophore, such asthrough a polymeric linker and/or spacer, and the antibody conjugate maybe detected directly. Antibody conjugates are described further in USPublication No. 2014/0147906 and U.S. Pat. Nos. 8,658,389; 8,686,122;8,618,265; 8,846,320; and 8,445,191.

FIGS. 15B and 15C illustrates one method for the multiplex detection oftargets where a tissue sample is contacted with one or more unmodifiedprimary antibodies and/or antibody conjugates (simultaneously orsequentially) (first stage, 220) and then subsequently contacted withone or more epitope-tagged antibodies (simultaneously or sequentially)(second stage, 250). Two stage multiplex assays are further illustratedin Examples 3, 4, 5, and 6 herein. The skilled artisan will recognizethat the first stage 220 and the second stage 250 may be reversed, suchthat the epitope-tagged antibodies are applied first to the issue samplefollowed by application of the unmodified antibodies and/or antibodyconjugates. The skilled artisan will also appreciate that appropriatenucleic acid probes may be substituted for the unmodified antibodiesand/or antibody conjugates such that the multiplex assay includes bothISH and IHC steps or stages (in any order).

In some embodiments, such as depicted in FIG. 15B, a first unmodifiedprimary antibody or antibody conjugate may be applied to a tissue sampleto form a first target-primary antibody complex (step 200). Next, firstdetection reagents specific to the unmodified primary antibody orantibody conjugate are applied to the tissue sample to detect the firsttarget-primary antibody complex (step 210). Dotted line 205 in FIG. 15Billustrates that steps 200 and 210 of first stage 220 may be repeatedone or more times to provide for the sequential multiplex detection ofmultiple, different targets within the tissue sample with unmodifiedprimary antibodies and/or antibody conjugates. For example, a secondunmodified primary antibody or antibody conjugate may be applied to thetissue sample to form a second target-primary antibody complex (200),followed by application of second detection reagents specific to thesecond unmodified primary antibody or antibody conjugate to detect thesecond target-primary antibody complex (210).

FIG. 15C represents an alternative method for the multiplex detection oftargets using a two stage method similar to that presented in FIG. 15B.In the method depicted in FIG. 15C, each of the unmodified antibodiesand/or antibody conjugates are simultaneously introduced to the tissuesample at step 260. Next, the sample is contacted with detectionreagents (e.g. anti-species antibodies of anti-label antibodies) at step270 to effectuate detection of the unmodified antibodies and/or antibodyconjugates. In an alternative embodiment, all of the unmodified primaryantibodies or antibody conjugates may be sequentially applied (step260), followed by sequential application of the respective anti-speciesantibodies (step 270).

The skilled artisan will appreciate that the detection reagents maycomprise anti-species antibodies specific to the utilized unmodifiedantibodies. Alternatively, the detection reagents may compriseanti-label antibodies specific to labels (e.g. haptens) conjugated tothe antibody conjugates. The skilled artisan will also appreciate thatthe anti-species or anti-label antibodies may comprise a detectablemoiety and, in embodiments where the detectable moiety is an enzyme,additional chromogenic substrates may be supplied with the first andsecond detection reagents.

Following the first stage of the multiplex assay 220 (FIG. 15B or 15C),a second stage 250 is performed, where the tissue sample issimultaneously contacted with a plurality of epitope-tagged antibodies(step 230), where each epitope-tagged antibody is specific for aparticular target, and where each epitope-tagged antibody comprises adifferent epitope tag. The epitope-tagged antibodies may be supplied tothe tissue sample as a “pool” or “cocktail” comprising each of theepitope-tagged antibodies needed for the particular assay. Eachepitope-tagged antibody will form a detectable target-epitope-taggedantibody complex with a specific target. Following the simultaneousapplication of the epitope-tagged antibodies (primary antibodies) (step230), anti-tag antibodies (secondary antibodies) are simultaneouslyapplied to the tissue sample (step 240), where each anti-tag antibody isspecific to one of the epitope-tagged antibodies applied, and where eachanti-tag antibody comprises a different detectable moiety. The anti-tagantibodies may be supplied to the tissue sample as a “pool” or“cocktail” comprising each of the anti-tag antibodies necessary fordetection of the target-epitope-tagged antibody complexes.

Following application of the anti-tag antibodies, in some embodimentsthe tissue sample may be stained with a counterstain (see, for example,Examples 3 through 6 which provide additional processing steps which maybe incorporated into any workflow). Signals from each of the detectablemoieties (e.g. from the anti-species, anti-label, and/or anti-tagantibodies) may be visualized or otherwise detected (e.g. simultaneouslyvisualized or detected).

As an example of a multiplex assay comprising both (i) unmodifiedantibodies and/or antibody conjugates, and (ii) epitope-taggedantibodies according to the present disclosure, a first antibodyconjugate comprising a hapten label (e.g. an anti-CD3 antibodyconjugated indirectly to a happen) is introduced to a tissue sample tofrom a target-antibody-conjugate complex. Simultaneously, an unmodifiedantibody (e.g. a rabbit anti-PDL1 antibody) is introduced to the tissuesample to form a target-unmodified-antibody complex. Next, detectionreagents are introduced (simultaneously or sequentially) to detect theformed target-antibody-conjugate complex (e.g. an anti-happen antibody)and the formed target-unmodified-antibody complex (e.g. a goatanti-rabbit antibody), where each of the detection reagents areconjugated to a different fluorophore.

In a second stage of the multiplex assay, a first epitope taggedantibody comprising a first epitope tag and specific to a first target(e.g. specific to CD68) is introduced to a tissue sample. In someembodiments, the first epitope-tagged antibody forms a detectable firsttarget-epitope-tagged antibody complex. Simultaneously, a second epitopetagged antibody comprising a second epitope tag and specific to a secondtarget (e.g. specific to FoxP3) is introduced to the sample to form asecond target-epitope-tagged antibody complex. Third, fourth, and nthadditional epitope-tagged antibodies specific to other targets (forming“n” target-epitope-tags antibody complexes) and having different epitopetags may be further introduced simultaneously with the first and secondepitope-tagged antibody antibodies.

After the epitope-tagged antibodies are deposited, they may be detected,either directly or indirectly depending, of course, on theirconfiguration. In some embodiments, anti-tag antibodies are introducedto enable detection of each of the target-epitope-tagged antibodycomplexes. In some embodiments, first, second, and nth detectionreagents are simultaneously introduced, where each of the first, second,and nth detection reagents are specific to the different epitope-taggedantibodies. In some embodiments, first, second, and nth detectionreagents are sequentially introduced, where each of the first, second,and nth detection reagents are specific to the different epitope-taggedantibodies. In some embodiments, the detection reagents are anti-tagantibodies that are specific to the different epitope tags of theepitope-tagged antibodies, and where the anti-tag antibodies are eachconjugated to a detectable moiety, e.g. a fluorophore of an enzyme. Insome embodiments, the detectable reagents are anti-tag antibodies eachconjugated to a fluorophore. In other embodiments, the detectablereagents are anti-tag antibodies each conjugated to an enzyme. In yetother embodiments, the detectable reagents are a combination of anti-tagantibodies conjugated to a fluorophore and anti-tag antibodiesconjugated to an enzyme. In those embodiments where the anti-tagantibodies are conjugated to an enzyme, substrates for the enzymes areprovided to effect detection (as noted previously herein).

In some embodiments, the multiplex detection method comprises the stepsof (i) contacting a biological sample with a first detection probecomprising an unmodified antibody to form a first target-antibodyconjugate complex; (ii) contacting the biological sample with a firstlabeling conjugate wherein the first labeling conjugate comprises afirst enzyme (where the first labeling conjugate is an anti-speciesantibody that specifically binds to the first unmodified antibody and isconfigured to label the target with an enzyme); (iii) contacting thebiological sample with a first signaling conjugate comprising a firstlatent reactive moiety and a first chromogenic moiety; (iv) inactivatingthe first enzyme, such as by contacting the sample with a first enzymeinactivation composition to substantially inactivate or completelyinactivate the first enzyme contained in the biological sample.Alternatively, the first detection probe comprising an unmodifiedantibody may be detected with an anti-species antibody coupled to afluorophore.

After the first enzyme is inactivated (optional), the multiplex methodfurther comprises the steps of (v) contacting a biological sample with asecond detection probe comprising an epitope-tagged antibody to form asecond target-antibody conjugate complex; (iv) contacting the biologicalsample with a second labeling conjugate wherein the second labelingconjugate comprises a second enzyme (where the second labeling conjugateis an anti-tag antibody that specifically binds to the second detectionprobe comprising an epitope-tagged antibody and is configured to labelthe target with an enzyme); (vii) contacting the biological sample witha second signaling conjugate comprising a second latent reactive moietyand a second chromogenic moiety; (viii) inactivating the second secondenzyme, such as by contacting the sample with a first enzymeinactivation composition to substantially inactivate or completelyinactivate the first enzyme contained in the biological sample.

After the second enzyme is inactivated, the method may be repeated suchthat additional detection probes (unmodified antibodies, antibodyconjugates, or epitope-tagged antibodies) may be introduced, along withadditional detection reagents, to effectuate detection of other targets.For example, a third detection probe comprising an epitope-taggedantibody expressing a different epitope tag may be introduced anddetected, such as with an anti-tag antibody conjugated to one of afluorophore or an enzyme. Following introduction of all of the detectionprobes and respective detection reagents or kits, the method furthercomprises the step of counterstaining the sample and/or detectingsignals (manually or via an automated method) from the first, second,and nth chromogenic moieties, wherein each of the first, second, and nthchromogenic moieties are each different.

Automation

The multiplex assays and methods may be semi-automated or automated. Forexample, the staining processes may be performed on a histochemicalstaining platform, such as an automated IHC/ISH slide stainer. AutomatedIHC/ISH slide stainers typically include at least: reservoirs of thevarious reagents used in the staining protocols, a reagent dispense unitin fluid communication with the reservoirs for dispensing reagent toonto a slide, a waste removal system for removing used reagents andother waste from the slide, and a control system that coordinates theactions of the reagent dispense unit and waste removal system. Inaddition to performing staining steps, many automated slide stainers canalso perform steps ancillary to staining (or are compatible withseparate systems that perform such ancillary steps), including: slidebaking (for adhering the sample to the slide), dewaxing (also referredto as deparaffinization), antigen retrieval, counterstaining,dehydration and clearing, and coverslipping. Prichard, Overview ofAutomated Immunohistochemistry, Arch Pathol Lab Med., Vol. 138, pp.1578-1582 (2014), incorporated herein by reference in its entirety,describes several specific examples of automated IHC/ISH slide stainersand their various features, including the intelliPATH (Biocare Medical),WAVE (Celerus Diagnostics), DAKO OMNIS and DAKO AUTOSTAINER LINK 48(Agilent Technologies), BENCHMARK (Ventana Medical Systems, Inc.), LeicaBOND, and Lab Vision Autostainer (Thermo Scientific) automated slidestainers. Additionally, Ventana Medical Systems, Inc. is the assignee ofa number of United States patents disclosing systems and methods forperforming automated analyses, including U.S. Pat. Nos. 5,650,327,5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S.Published Patent Application Nos. 20030211630 and 20040052685, each ofwhich is incorporated herein by reference in its entirety.Commercially-available staining units typically operate on one of thefollowing principles: (1) open individual slide staining, in whichslides are positioned horizontally and reagents are dispensed as apuddle on the surface of the slide containing a tissue sample (such asimplemented on the DAKO AUTOSTAINER Link 48 (Agilent Technologies) andintelliPATH (Biocare Medical) stainers); (2) liquid overlay technology,in which reagents are either covered with or dispensed through an inertfluid layer deposited over the sample (such as implemented on VENTANABenchMark and DISCOVERY stainers); (3) capillary gap staining, in whichthe slide surface is placed in proximity to another surface (which maybe another slide or a coverplate) to create a narrow gap, through whichcapillary forces draw up and keep liquid reagents in contact with thesamples (such as the staining principles used by DAKO TECHMATE, LeicaBOND, and DAKO OMNIS stainers). Some iterations of capillary gapstaining do not mix the fluids in the gap (such as on the DAKO TECHMATEand the Leica BOND). In variations of capillary gap staining termeddynamic gap staining, capillary forces are used to apply sample to theslide, and then the parallel surfaces are translated relative to oneanother to agitate the reagents during incubation to effect reagentmixing (such as the staining principles implemented on DAKO OMNIS slidestainers (Agilent)). In translating gap staining, a translatable head ispositioned over the slide. A lower surface of the head is spaced apartfrom the slide by a first gap sufficiently small to allow a meniscus ofliquid to form from liquid on the slide during translation of the slide.A mixing extension having a lateral dimension less than the width of aslide extends from the lower surface of the translatable head to definea second gap smaller than the first gap between the mixing extension andthe slide. During translation of the head, the lateral dimension of themixing extension is sufficient to generate lateral movement in theliquid on the slide in a direction generally extending from the secondgap to the first gap. See WO 2011-139978 A1. It has recently beenproposed to use inkjet technology to deposit reagents on slides. See WO2016-170008 A1. This list of staining technologies is not intended to becomprehensive, and any fully or semi-automated system for performingbiomarker staining may be incorporated into the histochemical stainingplatform.

Where a morphologically-stained sample is also desired, an automated H&Estaining platform may be used. Automated systems for performing H&Estaining typically operate on one of two staining principles: batchstaining (also referred to as “dip 'n dunk”) or individual slidestaining. Batch stainers generally use vats or baths of reagents inwhich many slides are immersed at the same time. Individual slidestainers, on the other hand, apply reagent directly to each slide, andno two slides share the same aliquot of reagent. Examples ofcommercially available H&E stainers include the VENTANA SYMPHONY(individual slide stainer) and VENTANA HE 600 (individual slide stainer)series H&E stainers from Roche; the Dako CoverStainer (batch stainer)from Agilent Technologies; the Leica ST4020 Small Linear Stainer (batchstainer), Leica ST5020 Multistainer (batch stainer), and the LeicaST5010 Autostainer XL series (batch stainer) H&E stainers from LeicaBiosystems Nussloch GmbH.

After the specimens are stained, the stained samples can be manuallyanalyzed on a microscope and/or digital images of the stained samplescan be collected for archiving and/or digital analysis. Digital imagescan be captured via a scanning platform such as a slide scanner that canscan the stained slides at 20×, 40×, or other magnifications to producehigh resolution whole-slide digital images. At a basic level, thetypical slide scanner includes at least: (1) a microscope with lensobjectives, (2) a light source (such as halogen, light emitting diode,white light, and/or multispectral light sources, depending on the dye),(3) robotics to move glass slides around or to move the optics aroundthe slide or both, (4) one or more digital cameras for image capture,(5) a computer and associated software to control the robotics and tomanipulate, manage, and view digital slides. Digital data at a number ofdifferent X-Y locations (and in some cases, at multiple Z planes) on theslide are captured by the camera's charge-coupled device (CCD), and theimages are joined together to form a composite image of the entirescanned surface. Common methods to accomplish this include:

(1) Tile based scanning, in which the slide stage or the optics aremoved in very small increments to capture square image frames, whichoverlap adjacent squares to a slight degree. The captured squares arethen automatically matched to one another to build the composite image;and

(2) Line-based scanning, in which the slide stage moves in a single axisduring acquisition to capture a number of composite image “strips.” Theimage strips can then be matched with one another to form the largercomposite image.

A detailed overview of various scanners (both fluorescent andbrightfield) can be found at Farahani et al., 5i Whole slide imaging inpathology: advantages, limitations, and emerging perspectives, Pathologyand Laboratory Medicine Int'l, Vol 7, p. 23-33 (June 2015), the contentof which is incorporated by reference in its entirety. Examples ofcommercially available slide scanners include: 3DHistech PANNORAMIC SCANII; DigiPath PATHSCOPE; Hamamatsu NANOZOOMER RS, HT, and XR; HuronTISSUESCOPE 4000, 4000XT, and HS; Leica SCANSCOPE AT, AT2, CS, FL, andSCN400; Mikroscan D2; Olympus VS120-SL; Omnyx VL4, and VL120;PerkinElmer LAMINA; Philips ULTRA-FAST SCANNER; Sakura FinetekVISIONTEK; Unic PRECICE 500, and PRECICE 600×; VENTANA ISCAN COREO andISCAN HT; and Zeiss AXIO SCAN.ZI. Other exemplary systems and featurescan be found in, for example, WO2011-049608) or in U.S. PatentApplication No. 61/533,114, filed on Sep. 9, 2011, entitled IMAGINGSYSTEMS, CASSETTES, AND METHODS OF USING THE SAME the content of whichis incorporated by reference in its entirety.

In some cases, the images may be analyzed on an image analysis system.Image analysis system may include one or more computing devices such asdesktop computers, laptop computers, tablets, smartphones, servers,application-specific computing devices, or any other type(s) ofelectronic device(s) capable of performing the techniques and operationsdescribed herein. In some embodiments, image analysis system may beimplemented as a single device. In other embodiments, image analysissystem may be implemented as a combination of two or more devicestogether achieving the various functionalities discussed herein. Forexample, image analysis system may include one or more server computersand a one or more client computers communicatively coupled to each othervia one of more local-area networks and/or wide-area networks such asthe Internet. The image analysis system typically includes at least amemory, a processor, and a display. Memory may include any combinationof any type of volatile or non-volatile memories, such as random-accessmemories (RAMs), read-only memories such as an Electrically-ErasableProgrammable Read-Only Memory (EEPROM), flash memories, hard drives,solid state drives, optical discs, and the like. It is appreciated thatmemory can be included in a single device, and can also be distributedacross two or more devices. Processor may include one or more processorsof any type, such as central processing units (CPUs), graphicsprocessing units (GPUs), special-purpose signal or image processors,field-programmable gate arrays (FPGAs), tensor processing units (TPUs),and so forth. It is appreciated that processor can be included in asingle device, and can also be distributed across two or more devices.Display may be implemented using any suitable technology, such as LCD,LED, OLED, TFT, Plasma, etc. In some implementations, display may be atouch-sensitive display (a touchscreen). Image analysis system alsotypically includes a software system stored on the memory comprising aset of instructions implementable on the processor, the instructionscomprising various image analysis tasks, such as object identification,stain intensity quantification, and the like. Exemplarycommercially-available software packages useful in implementing modulesas disclosed herein include VENTANA VIRTUOSO; Definiens TISSUE STUDIO,DEVELOPER XD, and IMAGE MINER; and Visopharm BIOTOPIX, ONCOTOPIX, andSTEREOTOPIX software packages.

Automated processes may also include a laboratory information system(LIS). LIS 130 typically performs one or more functions selected from:recording and tracking processes performed on samples and on slides andimages derived from the samples, instructing different components of theimmune context scoring system to perform specific processes on thesamples, slides, and/or images, and track information about specificreagents applied to samples and/or slides (such as lot numbers,expiration dates, volumes dispensed, etc.). LIS usually comprises atleast a database containing information about samples; labels associatedwith samples, slides, and/or image files (such as barcodes (including1-dimensional barcodes and 2-dimensional barcodes), radio frequencyidentification (RFID) tags, alpha-numeric codes affixed to the sample,and the like); and a communication device that reads the label on thesample or slide and/or communicates information about the slide betweenthe LIS and the other components of the immune context scoring system.Thus, for example, a communication device could be placed at each of asample processing station, automated histochemical stainer, H&E stainingplatform, and scanning platform. When the sample is initially processedinto sections, information about the sample (such as patient ID, sampletype, processes to be performed on the section(s)) may be entered intothe communication device, and a label is created for each sectiongenerated from the sample. At each subsequent station, the label isentered into the communication device (such as by scanning a barcode orRFID tag or by manually entering the alpha-numeric code), and thestation electronically communicates with the database to, for example,instruct the station or station operator to perform a specific processon the section and/or to record processes being performed on thesection. At scanning platform, the scanning platform may also encodeeach digital image with a computer-readable label or code thatcorrelates back to the section or sample from which the image isderived, such that when the image is sent to an image analysis system,image processing steps to be performed may be sent from the database ofLIS to the image analysis system and/or image processing steps performedon the image by image analysis system are recorded by database of LIS.Commercially available LIS systems useful in the present methods andsystems include, for example, VENTANA Vantage Workflow system (Roche).

Counterstaining

Counterstaining is a method of post-treating the samples after they havealready been stained with agents to detect one or more targets, suchthat their structures can be more readily visualized under a microscope.For example, a counterstain is optionally used prior to coverslipping torender the immunohistochemical stain more distinct. Counterstains shouldbe chosen that differ in color from a primary stain. Numerouscounterstains are well known, such as hematoxylin, eosin, methyl green,methylene blue, Giemsa, Alcian blue, and Nuclear Fast Red. DAPI(4′,6-diamidino-2-phenylindole) is a fluorescent stain that may be used.Counterstains may also be classified according to the structures towhich they bind and whether they are suitable for brightfield orfluorescent analysis, for example: brightfield nuclear counterstains,which include hematoxylin (stains from blue to violet), Methylene blue(stains blue), toluidine blue (stains nuclei deep blue andpolysaccharides pink to red), nuclear fast red (also called Kernechtrotdye, stains red), and methyl green (stains green); non-nuclearbrightfield stains, such as eosin (stains pink); fluorescent nuclearstains, including 4′, 6-diamino-2-phenylindole (DAPI, stains blue),propidium iodide (stains red), Hoechst stain (stains blue), nucleargreen DCS1 (stains green), nuclear yellow (Hoechst S769121, stainsyellow under neutral pH and stains blue under acidic pH), DRAQ5 (stainsred), DRAQ7 (stains red); fluorescent non-nuclear stains, such asfluorophore-labelled phalloidin, (stains filamentous actin, colordepends on conjugated fluorophore).

In some examples, more than one stain can be mixed together to producethe counterstain. This provides flexibility and the ability to choosestains. For example, a first stain, can be selected for the mixture thathas a particular attribute, but yet does not have a different desiredattribute. A second stain can be added to the mixture that displays themissing desired attribute. For example, toluidine blue, DAPI, andpontamine sky blue can be mixed together to form a counterstain.

Imaging

Certain aspects, or all, of the disclosed embodiments can be automated,and facilitated by computer analysis and/or image analysis system. Insome applications, precise color or fluorescence ratios are measured. Insome embodiments, light microscopy is utilized for image analysis.Certain disclosed embodiments involve acquiring digital images. This canbe done by coupling a digital camera to a microscope. Digital imagesobtained of stained samples are analyzed using image analysis software.Color or fluorescence can be measured in several different ways. Forexample, color can be measured as red, blue, and green values; hue,saturation, and intensity values; and/or by measuring a specificwavelength or range of wavelengths using a spectral imaging camera. Thesamples also can be evaluated qualitatively and semi-quantitatively.Qualitative assessment includes assessing the staining intensity,identifying the positively-staining cells and the intracellularcompartments involved in staining, and evaluating the overall sample orslide quality. Separate evaluations are performed on the test samplesand this analysis can include a comparison to known average values todetermine if the samples represent an abnormal state.

Samples and Targets

Samples include biological components and generally are suspected ofincluding one or more target molecules of interest. Target Molecules canbe on the surface of cells and the cells can be in a suspension, or in atissue section. Target molecules can also be intracellular and detectedupon cell lysis or penetration of the cell by a probe. One of ordinaryskill in the art will appreciate that the method of detecting targetmolecules in a sample will vary depending upon the type of sample andprobe being used. Methods of collecting and preparing samples are knownin the art.

Samples for use in the embodiments of the method and with thecomposition disclosed herein, such as a tissue or other biologicalsample, can be prepared using any method known in the art by of one ofordinary skill. The samples can be obtained from a subject for routinescreening or from a subject that is suspected of having a disorder, suchas a genetic abnormality, infection, or a neoplasia. The describedembodiments of the disclosed method can also be applied to samples thatdo not have genetic abnormalities, diseases, disorders, etc., referredto as “normal” samples. Such normal samples are useful, among otherthings, as controls for comparison to other samples. The samples can beanalyzed for many different purposes. For example, the samples can beused in a scientific study or for the diagnosis of a suspected malady,or as prognostic indicators for treatment success, survival etc.

Samples can include multiple targets that can be specifically bound by aprobe or reporter molecule. The targets can be nucleic acid sequences orproteins. In some examples, the target is a protein or nucleic acidmolecule from a pathogen, such as a virus, bacteria, or intracellularparasite, such as from a viral genome. For example, a target protein maybe produced from a target nucleic acid sequence associated with (e.g.,correlated with, causally implicated in, etc.) a disease.

The skilled artisan will appreciate that epitope-tagged antibodies maybe developed which are specific to any of the following targets:

In specific, non-limiting examples, a target protein is produced by atarget nucleic acid sequence (e.g., genomic target nucleic acidsequence) associated with a neoplasm (for example, a cancer). Numerouschromosome abnormalities (including translocations and otherrearrangements, amplification or deletion) have been identified inneoplastic cells, especially in cancer cells, such as B cell and T cellleukemias, lymphomas, breast cancer, colon cancer, neurological cancersand the like. Therefore, in some examples, at least a portion of thetarget molecule is produced by a nucleic acid sequence (e.g., genomictarget nucleic acid sequence) amplified or deleted in at least a subsetof cells in a sample.

In other examples, a target protein produced from a nucleic acidsequence (e.g., genomic target nucleic acid sequence) that is a tumorsuppressor gene that is deleted (lost) in malignant cells. For example,the p16 region (including D9S1749, D9S1747, p16(INK4A), p14(ARF),D9S1748, p15(INK4B), and D9S1752) located on chromosome 9p21 is deletedin certain bladder cancers. Chromosomal deletions involving the distalregion of the short arm of chromosome 1 (that encompasses, for example,SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322), and thepericentromeric region (e.g., 19p13-19q13) of chromosome 19 (thatencompasses, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, andGLTSCR1) are characteristic molecular features of certain types of solidtumors of the central nervous system.

Numerous other cytogenetic abnormalities that correlate with neoplastictransformation and/or growth are known to those of ordinary skill in theart. Target proteins that are produced by nucleic acid sequences (e.g.,genomic target nucleic acid sequences), which have been correlated withneoplastic transformation and which are useful in the disclosed methods,also include the EGFR gene (7p12; e.g., GENBANK™ Accession No.NC-000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21;e.g., GENBANK™ Accession No. NC-000008, nucleotides128817498-128822856), D5S271 (5p15.2), lipoprotein lipase (LPL) gene(8p22; e.g., GENBANK™ Accession No. NC-000008, nucleotides19841058-19869049), RB1 (13q14; e.g., GENBANK™ Accession No. NC-000013,nucleotides 47775912-47954023), p53 (17p13.1; e.g., GENBANK™ AccessionNo. NC-000017, complement, nucleotides 7512464-7531642)), N-MYC (2p24;e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides151835231-151854620), CHOP (12q13; e.g., GENBANK™ Accession No.NC-000012, complement, nucleotides 56196638-56200567), FUS (16p11.2;e.g., GENBANK.™ Accession No. NC-000016, nucleotides 31098954-31110601),FKHR (13p14; e.g., GENBANK™ Accession No. NC-000013, complement,nucleotides 40027817-40138734), as well as, for example, ALK (2p23;e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides29269144-29997936), Ig heavy chain, CCND1 (11q13; e.g., GENBANK™Accession No. NC-000011, nucleotides 69165054.69178423), BCL2 (18q21.3;e.g. GENBANK™ Accession No. NC-000018, complement, nucleotides58941559-59137593), BCL6 (3q27; e.g., GENBANK™ Accession No. NC-000003,complement, nucleotides 188921859-188946169), MALF1, AP1 (1p32-p31;e.g., GENBANK™ Accession No. NC-000001, complement, nucleotides59019051-59022373), TOP2A (17q21-q22; e.g., GENBANK™ Accession No.NC-000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3;e.g., GENBANK™ Accession No. NC-000021, complement, nucleotides41758351-41801948), ERG (21q22.3; e.g., GENBANK™ Accession No. N-000021mcomplement, nucleotides 38675671-38955488); ETV1 (7p21.3; e.g., GENBANK™Accession No. NC-000007, complement, nucleotides 13897379-13995289), EWS(22q12.2; e.g., GENBANK™ Accession No NC-000022, nucleotides27994271-28026505); FLI1 (11q24.1-q24.3; e.g., GENBANK™ Accession No.NC-000011, nucleotides 128069199-128187521), PAX3 (2q35-q37; e.g.,GENBANK™ Accession No. NC-000002, complement, nucleotides222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK™ Accession No.NC-000001, nucleotides 18830087-18935219), PTEN (10q23.3; e.g., GENBANK™Accession No. NC-000010, nucleotides 89613175-89716382), AKT2(19q13.1-q13.2; e.g., GENBANK™ Accession No. NC-000019, complement,nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK™ AccessionNo. NC-000001, complement, nucleotides 40133685-40140274), REL(2p13-p12; e.g., GENBANK™ Accession No. NC-000002, nucleotides60962256-61003682) and CSF1R (5q33-q35; e.g., GENBANK™ Accession No.NC-000005, complement, nucleotides 149413051-149473128).

EXAMPLES

The non-limiting examples presented herein each incorporate the use ofat least one epitope-tagged antibody. Applicants submit that theepitope-tagged antibodies disclosed herein are suitable for use in IHCassays, including multiplex IHC assays, as demonstrated in the followingexamples. Applicants also submit that the epitope-tagged antibodies maybe used in conjunction with unmodified antibodies or antibodyconjugates, as also shown in the following examples. Of course, asdetailed herein, the epitope-tagged antibodies may be used inconjunction with other detectable specific binding entities and may beutilized in assays with combine IHC and ISH.

In the embodiments which follow and those noted above, one or more washsteps may be performed, such as to remove unbound antibodies. In someembodiments, a wash step may be performed between each step of thestaining procedure to remove excess reagent, etc. Of course, the skilledartisan would understand the processes and procedures associated withany wash steps and be able to apply to those processes and procedures toremove any reagents, unbound antibodies, etc.

Example 1: 3-Plex Immunohistochemical Assay

Example 1 provides a multiplex immunohistochemical assay where threedifferent epitope-tagged antibodies were simultaneously applied to atissue sample (see FIG. 1). A first epitope-tagged antibody was specificfor CD68 (anti-CD68) and comprised the VSV epitope tag (a heavy chaincomprising four VSV epitope tags). A second epitope-tagged antibody wasspecific to FoxP3 (anti-FoxP3) and comprised the V5 epitope tag (a heavychain comprising five V5 epitope tags). A third epitope-tagged antibodywas specific to CD20 (anti-CD20) and comprised the HA epitope tag (aheavy chain comprising four H5 epitope tags). The epitope-taggedantibodies were applied as a “cocktail” comprise 2 μg/mL of eachepitope-tagged antibody in diluent 90039.

After the simultaneous application of the three epitope-taggedantibodies, three anti-tag antibodies were simultaneously supplied tothe tissue sample (see FIG. 1), where each anti-tag antibody wasspecific to a different epitope tag of the epitope-tagged antibodies. Afirst anti-VSV antibody was conjugated with Alexa 532 (producing an“orange” signal); a second anti-V5 antibody was conjugated with DyLight649 (producing a “green” signal); and a third anti-HA antibody wasconjugated with Alexa 594 (producing a “purple” signal). The anti-tagantibodies were applied as a “cocktail” comprise 5 μg/mL of eachanti-tag antibody in diluent 90040.

The following steps were undertaken for the 3-plex IHC assay (see Table2):

TABLE 2 Procedure Step Selection Deparaffinization Selected CellConditioning CC1, 64 min Tagged 1st Ab cocktail Incubate-32 min Blockingwith diluent 90040 32 min Anti-tag 2nd Ab cocktail Incubate-32 min DAPICounterstain 4 min ProLong Diamond anti-fade mounting

A complete protocol summary is provided below in Table 3:

TABLE 3  1 Paraffin (Selected)  2 Deparaffinization [Selected]  3 WarmupSlide to [72 Deg C.] from Medium Temperatures (Deparaffinization)  4Cell Conditioning (Selected)  5 Ultra cc 1 [Selected)  6 Warmup Slide to[100 Deg C.], and Incubate for 4 Minutes (Cell Conditioner #1)  7 CC1 8Min [Selected]  8 CC1 16 Min (Selected)  9 CC1 24 Min (Selected) 10 CC132 Min (Selected) 11 CC1 40 Min (Selected) 12 CC1 48 Min (Selected) 13CC1 56 Min (Selected) 14 CC1 64 Min (Selected) 15 1st Antibody ManualApplication [Selected] 16 Hand Apply (Primary Antibody). and Incubatefor [0 Hr 32 Min] 17 Blocker (Selected) 18 Apply One Drop of (OPTION 2)(2nd Option), and Incubate for (32 Minutes) 19 3rd Wash after Primary Ab(Selected) 20 2nd Antibody Manual Application [Selected] 21 Hand Apply(Secondary Antibody). and Incubate for (0 Hr 32 Min)

FIGS. 2A through 2D illustrate tissue samples stained with the 3-plexIHC assay noted above. FIG. 2A clearly shows signals from each of thefluorophores conjugated to the anti-tag antibodies, thus revealinglocations of each of the target-epitope-tagged antibody complexes,namely CD68-epitope-tagged antibody complexes (orange signals), theCD20-epitope-tagged antibody complexes (green signals), and theFoxp3-epitope-tagged antibody complexes (purple signals). The signalproduced from the DAPI counterstain appears blue in each of FIGS. 2Athrough 2D. FIG. 2B shows only the signals corresponding to the detectedCD68-epitope-tagged antibody complexes. FIG. 2C shows only the signalscorresponding to the detected Foxp3-epitope-tagged antibody complexes.FIG. 2D shows only signals corresponding to the detectedCD20-epitope-tagged antibody complexes. FIGS. 2A through 2D show thatthe epitope-tagged antibodies of the present disclosure were (1) capableof binding to CD68, CD20, and FoxP3, respectively; (2) capable of beingdetected by appropriate anti-tag antibodies; and (3) able to be appliedto a tissue sample simultaneously (e.g. as a cocktail of antibodies)without interfering with each other. Thus, the epitope-tagged antibodiesof the present disclosure were suitable for use in multiple-assays. Inaddition, the multiplex assay of the present example was able to becompleted in less than 4-hours. When compared to traditional multiplexassays, this represents an advancement in the art. In some embodiments,this panel may be used help immune profiling in haematological and solidtumors.

Example 2: 3-Plex Immunohistochemical Assay

Example 2 provides a multiplex immunohistochemical assay where threedifferent epitope-tagged antibodies were simultaneously applied to atissue sample (see FIG. 3). A first epitope-tagged antibody was specificfor CD68 (anti-CD68) and comprised the VSV epitope tag (a heavy chaincomprising four VSV epitope tags). A second epitope-tagged antibody wasspecific to FoxP3 (anti-FoxP3) and comprised the V5 epitope tag (a heavychain comprising five V5 epitope tags). A third epitope-tagged antibodywas specific to CD8 (anti-CD8) and comprised AU5 epitope tag (a heavychain comprising four AU5 epitope tags). The epitope-tagged antibodieswere applied as a “cocktail” comprise 2 μg/mL of each epitope-taggedantibody in diluent 90039.

After the simultaneous application of the three epitope-taggedantibodies, three anti-tag antibodies were simultaneously supplied tothe tissue sample (see FIG. 3), where each anti-tag antibody wasspecific to a different epitope tag of the epitope-tagged antibodies. Afirst anti-VSV antibody was conjugated with Alexa 532(JH) (producing an“orange” signal); a second anti-V5 antibody was conjugated with DyLight649 (producing a “green” signal); and a third anti-AU5 antibody wasconjugated with Alexa 594(JH) (producing a “purple” signal). Theanti-tag antibodies were applied as a “cocktail” comprise 5 μg/mL ofeach anti-tag antibody in diluent 90040.

The following steps were undertaken for the 3-plex IHC assay (see Table4):

TABLE 4 Procedure Step Selection Deparaffinization Selected CellConditioning CC1, 64 min Tagged 1st Ab cocktail Incubate-32 min Blockingwith diluent 90040 32 min Anti-tag 2nd Ab cocktail Incubate-32 min DAPICounterstain 4 min ProLong Diamond anti-fade mounting

A complete protocol summary is also provided at Table 3 above.

FIGS. 4A and 4B illustrate tissue samples stained with the 3-plex IHCassay noted above. FIGS. 4A and 4B clearly show signals from each of thefluorophores conjugated to the anti-tag antibodies, thus revealinglocations of each of the target-epitope-tagged antibody complexes,namely CD68-epitope-tagged antibody complexes (orange signals), theCD8-epitope-tagged antibody complexes (green signals), and theFoxP3-epitope-tagged antibody complexes (purple signals). The signalproduced from the DAPI counterstain appears blue in each of FIGS. 4A and4B. FIGS. 4A and 4B thus show that the epitope-tagged antibodies of thepresent disclosure were (1) capable of binding to CD68, CD8, and FoxP3respectively; (2) capable of being detected by appropriate anti-tagantibodies; and (3) able to be applied to a tissue sample simultaneously(e.g. as a cocktail of antibodies) without interfering with each other.Thus, the epitope-tagged antibodies of the present disclosure weresuitable for use in multiple-assays. In addition, the multiplex assay ofthe present example was able to be completed in less than 4-hours. Whencompared to traditional multiplex assays, this again represents anadvancement in the art. This panel of immune markers help immuneprofiling in hematological and solid tumors.

Example 3: 4-Plex Immunohistochemical Assay Using Combined Anti-Speciesand Anti-Tag Antibodies

Example 3 provides a multiplex immunohistochemical assay where fourdifferent epitope-tagged antibodies were applied to a tissue sample (seeFIG. 5) in two stages. First, an unmodified antibody, namely ananti-Pan-CK antibody, was contacted with the tissue sample. Followingapplication of the anti-Pan-CK antibody, a goat anti-mouse-Alexa 488antibody was applied to detect the target-anti-Pan-CK antibody complex(Goat anti-mouse-Alexa 488 (2 μg/mL) in diluent 90040).

A second stage was then conducted where three epitope-tagged antibodieswere simultaneously supplied to the tissue sample (see FIG. 5). In thissecond stage, a first epitope-tagged antibody was specific for CD68(anti-CD68) and comprised the VSV epitope tag (a heavy chain comprisingfour VSV epitope tags). A second epitope-tagged antibody was specific toFoxP3 (anti-FoxP3) and comprised the V5 epitope tag (a heavy chaincomprising five V5 epitope tags). A third epitope-tagged antibody wasspecific to CD8 (anti-CD8) and comprised the AU5 epitope tag (a heavychain comprising four AU5 epitope tags). The epitope-tagged antibodieswere applied as a “cocktail” comprise 2 μg/mL of each epitope-taggedantibody in diluent 90039.

After the simultaneous application of the three epitope-taggedantibodies, three anti-tag antibodies were simultaneously supplied tothe tissue sample (see FIG. 5), where each anti-tag antibody wasspecific to a different epitope tag of the epitope-tagged antibodies. Afirst anti-VSV antibody was conjugated with Alexa 532(JH); a secondanti-V5 antibody was conjugated with DyLight 649; and a third anti-AU5antibody was conjugated with Alexa 594(JH). The anti-tag antibodies wereapplied as a “cocktail” comprise 5 μg/mL of each anti-tag antibody indiluent 90040.

The following steps were undertaken for the 4-plex IHC assay (see Table5):

TABLE 5 Procedure Step Selection Deparaffinization Selected CellConditioning CC1, 64 min Mouse anti-pan-CK 32 min Goat anti-mouse-Alexa488 32 min Blocking reagent 90040 32 min Tagged 1st Ab cocktailIncubate-32 min Blocking with diluent 90040 32 min Anti-tag 2nd Abcocktail Incubate-32 min DAPI Counterstain 4 min ProLong Diamondanti-fade mounting

A complete protocol summary is provided at Tables 6B, 6C, and 6D.

FIGS. 6A through 6F illustrate tissue samples stained with the 4-plexIHC assay noted above. FIG. 6A clearly shows signals from each of thefluorophores conjugated to either the anti-species antibody or anti-tagantibodies, thus revealing locations of each of the target-antibodycomplexes, namely the Pan-CK-antibody complexes (“green” signals), theCD68-epitope-tagged antibody complexes (“orange” signals), theCD8-epitope-tagged antibody complexes (“red” signals), and theFoxp3-epitope-tagged antibody complexes (“cyan” signals). The signalproduced from the DAPI counterstain appears blue in each of FIGS. 6Athrough 6F. FIG. 6B shows only the signals corresponding to the DAPIcounterstain. FIG. 6C shows only the signals corresponding to thedetected CD8-epitope-tagged antibody complexes. FIG. 6D shows only thesignals corresponding to the detected CD68-epitope-tagged antibodycomplexes. FIG. 6E shows only signals corresponding to the detectedFoxP3-epitope-tagged antibody complexes. FIG. 6F shows only signalscorresponding to the detected Pan-CK staining. FIGS. 6A through 6E showthat the epitope-tagged antibodies of the present disclosure were (1)capable of binding to CD68, CD8, and FoxP3, respectively; (2) capable ofbeing detected by appropriate anti-tag antibodies; and (3) able to beapplied to a tissue sample simultaneously (e.g. as a cocktail ofantibodies) without interfering with each other. FIG. 6A alsoillustrates that the epitope-tagged antibodies may be combined in anassay with unmodified antibodies or antibody conjugates, such as mouseanti-Pan-CK antibodies, and that such a combination allows for thedetection of all fluorophores conjugated to anti-species or anti-tagantibodies. In addition, the multiplex assay of the present example wasable to be completed within 4 hours. When compared to traditionalmultiplex assays, this represents an advancement in the art. This panelof markers may be useful to show the expression and distribution of theabove-mentioned immune cell markers and epithelial cell marker within atumor region.

Example 4: 4-Plex Immunohistochemical Assay Using Combined Anti-Speciesand Anti-Tag Antibodies

Example 4 provides a multiplex immunohistochemical assay where fourdifferent epitope-tagged antibodies were applied to a tissue sample (seeFIG. 7) in two stages. First, an unmodified antibody, namely ananti-Pan-CK antibody, was contacted with the tissue sample. Followingapplication of the anti-Pan-CK antibody, a goat anti-mouse-Alexa 488antibody was applied to detect the target-anti-Pan-CK antibody complex(Goat anti-mouse-Alexa 488 (2 μg/mL) in diluent 90040).

A second stage was then conducted where three epitope-tagged antibodieswere simultaneously supplied to the tissue sample (see FIG. 7). In thissecond stage, a first epitope-tagged antibody was specific for CD68(anti-CD68) and comprised the VSV epitope tag (a heavy chain comprisingfour VSV epitope tags). A second epitope-tagged antibody was specific toFoxP3 (anti-FoxP3) and comprised the V5 epitope tag (a heavy chaincomprising five V5 epitope tags). A third epitope-tagged antibody wasspecific to CD20 (anti-CD20) and comprised the HA epitope tag (a heavychain comprising four HA epitope tags). The epitope-tagged antibodieswere applied as a “cocktail” comprise 2 μg/mL of each epitope-taggedantibody in diluent 90039.

After the simultaneous application of the three epitope-taggedantibodies, three anti-tag antibodies were simultaneously supplied tothe tissue sample (see FIG. 7), where each anti-tag antibody wasspecific to a different epitope tag of the epitope-tagged antibodies. Afirst anti-VSV antibody was conjugated with Alexa 532(JH); a secondanti-V5 antibody was conjugated with DyLight 649; and a third anti-AU5antibody was conjugated with Coumarin (JH). The anti-tag antibodies wereapplied as a “cocktail” comprise 5 μg/mL of each anti-tag antibody indiluent 90040.

The following steps were undertaken for the 4-plex IHC assay (see Table6A):

TABLE 6A Procedure Step Selection Deparaffinization Selected CellConditioning CC1, 64 min Mouse anti-pan-CK 32 min Goat anti-mouse-Alexa488 32 min Blocking reagent 90040 32 min Tagged 1st Ab cocktailIncubate-32 min Blocking with diluent 90040 32 min Anti-tag 2nd Abcocktail Incubate-32 min DAPI Counterstain 4 min ProLong Diamondanti-fade mounting

A complete protocol summary is provided at Tables 6B, 6C, and 6D:

TABLE 6B  1 Enable Mixers  2 Disable Mixers  3 [72 C. is the standardtemperature]  4 Warm up Slide m (72 Deg C.) from Medium Temperatures(Deparaffinization)  5 Incubate for 4 minutes  6 Apply EZPrep VolumeAdjust  7 Rinse Slide With EZ Prep  8 Apply EZPrep Volume Adjust  9Apply Coverslip 10 Rinse Slide With EZ Prep 11 Apply EZPrep VolumeAdjust 12 Apply Coverslip 13 Enable Mixers 14 Disable Slide Heater 15Pause Point I Landing Zone 1 16 Rinse Slide With EZ Prep 17 Apply longCell Conditioner #1 18 Apply CC Coverslip Long 19 [100 C. is thestandard temperature] 20 Warmly Slide ID (100 Deg C. and Incubate for 4Minutes (Cell Conditioner #1) 21 Incubate for 4 Minutes 22 Incubate for8 minutes 23 Apply Cell conditioner #1 24 Apply CC Medium Coverslip NoBB 25 Incubate for 8 minutes 26 Incubate for 8 minutes 27 Apply Cellconditioner #1 28 Apply CC Medium Coverslip No BB 29 Incubate for 8minutes 30 incubate for 8 minutes 31 apply Cell conditioner #1 32 ApplyCC medium Coverslip No BB 33 Incubate for 8 minutes 34 Apply Cellconditioner #1 35 Apply CC Medium Coverslip No BB 36 Apply Cellconditioner #1 37 Apply CC Medium Coverslip No BB 38 Apply Cellconditioner #1 39 Apply CC Medium Coverslip No BB 40 Disable SlideHeater 41 Apply Cell conditioner #1 42 Apply CC Medium Coverslip No BB43 Rinse slide with Reaction Buffer 44 Adjust slide volume with reactionbuffer 45 Apply Coverslip

TABLE 6C 46 Rinse Slide With Reaction Buffer 47 Aqus1 Side Volume WithReaction Buffer 48 Apply Coverslip 49 Pause Point Landing Zone 50 WarmupSlide ID 36 Deg C. 51 Rinse Slide with reaction Buffer 52 Adjust SlideVolume with reaction Buffer 53 Apply one drop of anti-pan keratin(antibody), apply coverslip, and incubate for 0 hr and 16 min 54 Rinseslide with reaction buffer 55 Adjust Slide volume with reaction buffer56 Apply Coverslip 57 Rinse slide with reaction buffer 58 Adjust Slidevolume with reaction buffer 59 Apply Coverslip 60 Apply one drop ofPretreatment #1 and incubate for 32 minutes 61 Rinse slide with reactionbuffer 62 Adjust Slide volume with reaction buffer 63 [blocking buffer]64 Apply Coverslip 65 Rinse slide with reaction buffer 66 Adjust Slidevolume with reaction buffer 67 Apply Coverslip 68 Hand Apply (SecondaryAntibody), and incubate for 32 minutes 69 Rinse slide with reactionbuffer 70 Adjust Slide volume with reaction buffer 71 Apply Coverslip 72Rinse Slide With Reaction Buffer 73 Adjust Slide volume with reactionbuffer 74 Apply Coverslip 75 Rinse Slide With Reaction Buffer 76 AdjustSlide volume with reaction buffer 77 Apply Cover-slip 78 Hand Apply{Primary antibody). and Incubate for 32 minutes 79 (Ab w/Tags) 80 Rinseslide with reaction buffer 81 Adjust Slide volume with reaction buffer82 Apply Coverslip 83 Rinse slide with reaction buffer 84 Adjust Slidevolume with reaction buffer 85 Apply Coverslip 86 Apply One Drop of[OPTION 2] (2nd option) and Incubate for 32 minutes 87 Rinse Slide WithReaction Buffer 88 Adjust Slide volume with reaction buffer 89 [BlockingBuffer] 90 Apply Coverslip

TABLE 6D 91 Rinse slide with reaction buffer 92 Adjust Slide volume withreaction buffer 93 Apply Coverslip 94 Hand Apply (Secondary Antibody),and incubate for 32 minutes 95 Rinse slide with reaction buffer 96Adjust Slide volume with reaction buffer 97 Apply Coverslip 98 RinseSlide With Reaction Buffer 99 Adjust Slide volume with reaction buffer100  Apply Coverslip

FIGS. 8A and 8B illustrate tissue samples stained with the 4-plex IHCassay noted above. FIG. 8A provides four images, each image showingsignals corresponding to detected Pan-CK, FoxP3, CD68, and CD20. FIG. 8Bprovides an image where each of the signals corresponding to detectedPan-CK, FoxP3, CD68, and CD20, the image derived from a tissue sampleonto which all four of the above-identified antibodies were applied.Once again, FIGS. 8A and 8B show that the epitope-tagged antibodies ofthe present disclosure were (1) capable of binding to CD68, CD20, andFoxP3, respectively; (2) capable of being detected by appropriateanti-tag antibodies; and (3) able to be applied to a tissue samplesimultaneously (e.g. as a cocktail of antibodies) without interferingwith each other. FIG. 8B also illustrates that the epitope-taggedantibodies may be combined in an assay with unmodified antibodies orantibody conjugates, such as mouse anti-Pan-CK antibodies and that sucha combination allows for the detection of all fluorophores conjugated toanti-species or anti-tag antibodies. In addition, the multiplex assay ofthe present example was able to be completed within 4 hours. Whencompared to traditional multiplex assays, this represents an advancementin the art. This panel of markers may be useful to show the expressionand distribution of the above-mentioned immune cell markers andepithelial cell marker for tumor region.

Example 5: 4-Plex Immunohistochemical Assay Using Combined Anti-Speciesand Anti-Tag Antibodies

Example 5 provides a multiplex immunohistochemical assay where fourdifferent epitope-tagged antibodies were applied to a tissue sample (seeFIG. 9) in two stages. As compared with Examples 3 and 5, Example 5provides two antibodies in a first stage and another two epitope-taggedantibodies in a second stage.

With regard to the first stage depicted in FIG. 9, a first unmodifiedantibody, namely an anti-Pan-CK antibody, was contacted with the tissuesample. Simultaneously or subsequent to the application of the firstunmodified antibody, a second unmodified antibody, namely ananti-PDL1(SP142) antibody, was contacted with the tissue sample.Following application of the first and second unmodified antibodies, agoat anti-mouse-Alexa 488 antibody was applied to detect thetarget-anti-Pan-CK antibody complexes (Goat anti-mouse-Alexa 488 (2μg/mL) in diluent 90040) and a goat anti-rabbit-Alexa 594 antibody wasapplied to detect the target-anti-PDL1(SP142) antibody complexes (Goatanti-Rabbit-Alexa 594 (2 μg/ml) in 90040).

A second stage was then conducted where two epitope-tagged antibodieswere simultaneously supplied to the tissue sample (see FIG. 9). In thissecond stage, a first epitope-tagged antibody was specific for CD20(anti-CD20) and comprised the HA epitope tag (a heavy chain comprisingfour HA epitope tags). A second epitope-tagged antibody was specific toCD68 (anti-CD68) and comprised the VSV epitope tag (a heavy chaincomprising four VSV epitope tags). The epitope-tagged antibodies wereapplied as a “cocktail” comprise 2 μg/mL of each epitope-tagged antibodyin diluent 90039.

After the simultaneous application of the two epitope-tagged antibodies,two anti-tag antibodies were simultaneously supplied to the tissuesample (see FIG. 9), where each anti-tag antibody was specific to adifferent epitope tag of the epitope-tagged antibodies. A first anti-HAantibody was conjugated with Coumarin; and a second anti-VSV antibodywas conjugated with Alexa 532. The anti-tag antibodies were applied as a“cocktail” comprise 5 μg/mL of each anti-tag antibody in diluent 90040.

The following steps were undertaken for the 4-plex IHC assay (see Table7):

TABLE 7 Procedure Step Selection Deparaffinization Selected CellConditioning CC1, 64 min Mouse anti-pan-CK and Rabbit anti- 32 min PDL1Blocking reagent 90040 32 min Goat anti-mouse-Alexa 488 and Goat 32 minanti-Rabbit Alexa 594 Negative Control Rabbit IgG 32 min Blocking withdiluent 90040 32 min Tagged 1st Ab cocktail Incubate-32 min Blockingwith diluent 90040 32 min Anti-tag 2nd Ab cocktail Incubate-32 min DAP1Counterstain 4 min ProLong Diamond anti-fade mounting

A complete protocol summary is provided at Table 8:

TABLE 8 1 Paraffin [Selected] 2 Deparaffinization (Selected] 3 WarmupSlide to [72 Deg C.] from Medium Temperatures (Deparaffinization) 4 CellConditioning (Selected] 5 Ultra CC1 (Selected] 6 Warmup Slide to (100Deg C.], and Incubate for 4 Minutes (Cell Conditioner #1) 7 CC1 8 Min(Selected) 8 CC1 16 Min (Selected] 9 CC1 24 Min (Selected] 10 CC1 32 Min(Selected) 11 CC1 40 Min (Selected) 12 CC1 48 Min (Selected] 13 CC1 56Min (Selected) 14 CC1 64 Min (Selected 15 Research fork #1 (Selected] 16Hand Apply (Antibody) and Incubate for [0 Hr 16 Min] 17 Blocker[Selected] 18 Apply One Drop of [OPTION 2] (Option 1), and Incubate for[32 Minutes] 19 3rd wash after Primary Ab (Selected] 20 Research Fork #2(Selected] 21 Hand Apply (Secondary Antibody), and Incubate for [0 Hr 32Min] 22 Research Fork #9 (Selected) 23 Apply Three Drops of (NEG CTL RbtIg] (Antibody) and Incubate for (32 Minutes] 24 Research Fork #3(Selected] 25 Apply One Drop of [OPTION 2] (Option 2) and Incubate for(32 Minutes] 26 Research Fork #4 (Selected] 27 Hand Apply (PrimaryAntibody), and Incubate for (0 Hr 32 Min] 28 Research Fork #5 (Selected]29 Research Fork #6 (Selected] 30 Apply One Drop of (OPTION 2] (Option3) and Incubate for 32 Minutes] 31 Research Fork #7 (Selected] 32Research Fork #8 (Selected] 33 Counterstain Options [Selected] 34 ApplyOne Drop of [HEMATOXYLIN II] (Counterstain), Apply Coverslip, andIncubate for (4 Minutes]

FIGS. 10A through 10G illustrate tissue samples stained with the 4-plexIHC assay noted above. FIG. 10A provides images showing DAPI staining,PDL1 staining, CD20 staining, and CD68 staining, each individually. FIG.10B provides an image showing signals corresponding to DAPI staining,PDL1 staining, CD20 staining, CD68 staining, and Pan-CK staining. FIG.10C provides individual images showing each of Pan-CK staining and PDL1staining; and an image showing signals corresponding to Pan-CK stainingand PDL1 staining. FIG. 10C illustrates that two unmodified antibodiesraised from different species may be combined and detected usinganti-species antibodies, where the signals allow to show tumor region(Pan-CK positive tumor epithelial cells) and PDL1 expressing tumor cells(co-localization of Pan-CK and PDL1) or PDL1 expressing immune cells.FIGS. 10D and 10E each provide images showing CD68 and PDL1 co-staining,where macrophages (CD68 positive) express PDL1, a known phenomenon. Insome embodiments, this particular assay may be used to identify tumorinfiltrating lymphocytes. FIGS. 10D and 10E thus illustrates thatunmodified antibodies and epitope-tagged antibodies may be successfullycombined, across two stages of application, and that each of theantibodies may be detected by anti-species or anti-tag antibodies. FIGS.10D and 10E also illustrate the co-localization of cell membranesexpressing both PDL1 and having the CD68 marker. FIGS. 10F and 10Gillustrate a tissue sample stained with PDL1, Pan-CK, CD68, and CD20.

Once again, FIGS. 10A through 10G show that the epitope-taggedantibodies of the present disclosure were (1) capable of binding to CD20and CD68, respectively; (2) capable of being detected by appropriateanti-tag antibodies, and (3) able to be applied to a tissue samplesimultaneously (e.g. as a cocktail of antibodies) without interferingwith each other. FIGS. 10A through 10G also illustrate that theepitope-tagged antibodies may be combined in an assay with twounmodified antibodies or antibody conjugates, such as mouse anti-Pan-CKantibodies or rabbit anti-PDL1(SP142) antibodies and that such acombination allows for the detection of all fluorophores conjugated toanti-species or anti-tag antibodies. In addition, the multiplex assay ofthe present example was able to be completed within 4 hours. Whencompared to traditional multiplex assays, this represents an advancementin the art. This panel of markers allow for the identification of theexpression of PD-L1 in tumor cells and infiltrating immune cells intumor microenvironment on patients with anti-PD-L1 treatment].

Example 6: 4-Plex Immunohistochemical Assay Using Combined Anti-Speciesand Anti-Tag Antibodies

Example 6 provides a multiplex immunohistochemical assay where fourdifferent epitope-tagged antibodies were applied to a tissue sample (seeFIG. 11) in two stages. As compared with Examples 3 and 5, Example 5provides two antibodies in a first stage and another two epitope-taggedantibodies in a second stage.

With regard to the first stage depicted in FIG. 11, a first unmodifiedantibody, namely an anti-Pan-CK antibody, was contacted with the tissuesample. Simultaneously or subsequent to the application of the firstunmodified antibody, a second unmodified antibody, namely ananti-CD3(SP162) antibody, was contacted with the tissue sample.Following application of the first and second unmodified antibodies, agoal anti-mouse-Alexa 488 antibody was applied to detect thetarget-anti-Pan-CK antibody complexes (Goat anti-mouse-Alexa 488 (2μg/mL) in diluent 90040) and a goat anti-rabbit-Alexa 594 antibody wasapplied to detect the target-anti-PDL1(SP142) antibody complexes (Goatanti-Rabbit-Alexa 594 (2 ug/ml) in 90040).

A second stage was then conducted where two epitope-tagged antibodieswere simultaneously supplied to the tissue sample (see FIG. 11). In thissecond stage, a first epitope-tagged antibody was specific for CD20(anti-CD20) and comprised the HA epitope tag (a heavy chain comprisingfour HA epitope tags). A second epitope-tagged antibody was specific toCD68 (anti-CD68) and comprised the VSV epitope tag (a heavy chaincomprising four VSV epitope tags). The epitope-tagged antibodies wereapplied as a “cocktail” comprise 2 μg/mL of each epitope-tagged antibodyin diluent 90039.

After the simultaneous application of the two epitope-tagged antibodies,two anti-tag antibodies were simultaneously supplied to the tissuesample (see FIG. 11), where each anti-tag antibody was specific to adifferent epitope tag of the epitope-tagged antibodies. A first anti-HAantibody was conjugated with Coumarin; and a second anti-VSV antibodywas conjugated with Alexa 532. The anti-tag antibodies were applied as a“cocktail” comprise 5 μg/mL of each anti-tag antibody in diluent 90040.

The following steps were undertaken for the 4-plex IHC assay (Table 9):

TABLE 9 Procedure Step Selection Deparaffinization Selected CellConditioning CC1, 64 min Mouse anti-pan-CK and Rabbit anti-CD3 32 minBlocking reagent 90040 32 min Goat anti-mouse-Alexa 488 and Goat 32 minanti-Rabbit Alexa 594 Negative Control Rabbit IgG 32 min Blocking withdiluent 90040 32 min Tagged 1st Ab cocktail Incubate-32 min Blockingwith diluent 90040 32 min Anti-tag 2nd Ab cocktail Incubate-32 min DAPICounterstain  4 min ProLong Diamond anti-fade mounting

A complete protocol summary is provided at Table 8.

FIGS. 12A through 12D illustrate tissue samples stained with the 4-plexIHC assay noted above. FIG. 12A provides images showing DAPI staining,CD3 staining, PanCK staining, and CD68 staining. FIG. 12B provides animage showing signals corresponding to CD20 staining and CD3 staining,which represent the two distinct B cell and T cell populations. FIG. 12Billustrates that unmodified antibodies (anti-CD3) and epitope-taggedantibodies (anti-CD68) may be successfully combined, across two stagesof application, and that each of the antibodies may be detected byanti-species or anti-tag antibodies, respectively. FIGS. 12C and 12Dillustrate a tissue sample stained with CD3, Pan-CK, CD68, and CD20.

FIGS. 12A through 12D show that the epitope-tagged antibodies of thepresent disclosure were (1) capable of binding to CD20 and CD68,respectively; (2) capable of being detected by appropriate anti-tagantibodies; and (3) able to be applied to a tissue sample simultaneously(e.g., as a cocktail of antibodies) without interfering with each other.FIGS. 12A through 12D also illustrate that the epitope-tagged antibodiesmay be combined in an assay with two unmodified antibodies or antibodyconjugates, such as mouse anti-Pan-CK antibodies ofrabbit-anti-CD3(SP162) antibodies and that such a combination allows forthe detection of all fluorophores conjugated to anti-species or anti-tagantibodies. In addition, the multiplex assay of the present example wasable to be completed within 4 hours. When compared to traditionalmultiplex assays, this represents an advancement in the art. This panelof markers illustrates the immune cell markers and tumor cell marker,which help illustrate an immune profile in tumor microenvironment.

Example 7: 5-Plex Immunohistochemical Assay

Example 7 provides a multiplex immunohistochemical assay where fivedifferent epitope-tagged antibodies were simultaneously applied to atissue sample (see FIG. 13). A first epitope-tagged antibody wasspecific for CD3 (anti-CD3) and comprised the E epitope tag (a heavychain comprising four E epitope tags). A second epitope-tagged antibodywas specific to CD8 (anti-CD8) and comprised the E2 epitope tag (a heavychain comprising four E2 epitope tags). A third epitope-tagged antibodywas specific to CD20 (anti-CD20) and comprised the HA epitope tag (aheavy chain comprising four HA epitope tags). A fourth epitope-taggedantibody was specific to CD68 (anti-CD68) and comprised the VSV epitopetag (a heavy chain comprising four VSV epitope tags). A fifthepitope-tagged antibody was specific to FoxP3 (anti-FoxP3) and comprisedthe V5 epitope tag (a heavy chain comprising five V5 epitope tags). Theepitope-tagged antibodies were applied as a “cocktail” comprise 2 μg/mLof each epitope-tagged antibody in diluent 90039.

After the simultaneous application of the three epitope-taggedantibodies, five anti-tag antibodies were simultaneously supplied to thetissue sample (see FIG. 13), where each anti-tag antibody was specificto a different epitope tag of the epitope-tagged antibodies. A firstanti-E antibody was conjugated with Alexa 488; a second anti-E2 antibodywas conjugated with Alexa 594; a third anti-HA antibody was conjugatedwith Courmarin; a fourth anti-VSV antibody was conjugated to Alexa 532;and a fifth anti-V5 antibody was conjugated to Alexa 647. The anti-tagantibodies were applied as a “cocktail” comprise 5 μg/mL of eachanti-tag antibody in diluent 90040.

The following steps were undertaken for the 3-plex IHC assay (Table 10):

TABLE 10 Procedure Step Selection Deparaffinization Selected CellConditioning CC1, 64 min Tagged 1st Ab cocktail Incubate-32 min Blockingwith diluent 90040 32 min Anti-tag 2nd Ab cocktail Incubate-32 min DAPICounterstain  4 min ProLong Diamond anti-fade mounting

A complete protocol summary is provided at Table 3.

FIG. 14A provides an image showing showing signals corresponding toDAPI, CD20, CD3, CD8, CD68, and FoxP3 staining. FIG. 14B provides animage showing DAPI, CD3 and CD8 staining. Notably, FIG. 14B illustrateslocations where the CD3 and CD8 signals are co-registered (in thepresence of the DAPI counterstain) such that the CD3 white signals andCD8 red signals overlap to provide co-registered magenta signals.Similarly, FIG. 14C illustrates co-registration of CD3 (green) and CD8(red) signals, without the presence of the DAPI counterstain.

FIG. 14D provides an image showing DAPI, CD8 (cytotoxic T cell marker)and FoxP3 (regulatory T cell marker) staining, where no co-registrationis observed. FIG. 14E provides an image showing CD8 (cytotoxic T cellmarker) and FoxP3 (regulatory T cell marker) staining, where noco-registration is observed. FIG. 14F provides an image showing CD3 (Tcell marker) and FoxP3 (regulatory T cell marker) staining, whereco-registration is observed. FIG. 14G provides an image showing CD68 andFoxP3 staining; while FIG. 14H provides an image showing CD20 (B cellmarker) and CD3 (T cell marker) staining. FIG. 14I provides an imageshowing CD3, FoxP3, and CD8 staining, where CD8 (cytotoxic T cellmarker) and FoxP3 (regulatory T cell marker) are co-localized to CD3 (Tcell marker). FIG. 14J provides an image showing CD3, FoxP3, CD8, andCD68 staining. These staining patterns illustrate different populationsof immune cells and their relationship to one another.

FIGS. 14A through 14I show that the five epitope-tagged antibodies ofthe present disclosure were (1) capable of binding to CD20, CD3, CD8,CD68, and FoxP3; (2) capable of being detected by appropriate anti-tagantibodies; and (3) able to be applied to a tissue sample simultaneously(e.g. as a cocktail of antibodies) without interfering with each other.The 5-plex MIHC assay was able to be completed in less than four hours.This assay is useful for immune profiling in in hematological and solidtumors.

Example 8: Evaluation of HER2/neu (4B5) Antibody—V5 Peptide Conjugateson HER2+ 4nl Cell Line Models and Breast Cancer Tissue

This example demonstrates the visualization of HER2/neu (4B5) antibodyV5 peptide conjugates on HER2+ 4nl cell line models and HER2+ breastcancer tissue. Slides containing tonsil tissue sections were developedusing a standard protocol for an automated stainer [BenchMark® XT,Ventana Medical Systems, Inc, (VMSI) Tucson, Ariz.]. A typical automatedprotocol is as follows: The paraffin-coated tissue on the slides washeated to 75° C. for 4 minutes and treated once with EZPrep (VMSI#950-102), volume adjusted at 75° C. before application of the LiquidCover Slip (LCS, VMSI #650-010). After the slide was incubated for 4minutes at 75° C., the slide was rinsed and EZPrep volume was adjusted,followed with LCS to deparaffinize the tissue (process repeated threetimes—4 total cycles). The slides were cooled to 37° C. and incubatedfor 4 minutes. The slides were thoroughly rinsed with cell conditionsolution (CCI, VMSI #950-124), followed by application of LCS. Theslides were heated to 95° C. for 8 minutes. The slides were then heatedto 100° C. and incubated for 8 minutes. The slides were rinsed with CCIfollowed by application of LCS and incubated for 8 minutes at 100° C.Every 4 minutes, for 16 minutes, CCI and LCS were applied in order toprevent slide drying.

The slides were cooled to 37° C. and rinsed twice with reaction buffer(VMSI #950-300), 100 μL of UV Inhibitor (a component of the VMSI ultraView DAB Detection Kit #760-500) was applied to the slide and incubatedfor 4 minutes. The slides were rinsed once with reaction buffer beforethe application of 100 μL of HER2/neu antibody-VS peptide conjugate (1μg/mL) in Ventana Antibody Diluent with Casein (VMSI #760-219) for 16minutes at 37° C. The slides were rinsed 3 times with reaction bufferbefore the addition of 100 μL of Pierce MsAntiV5 Antibody (CloneEIO/V4RR, Pierce #MAS-15253) at 1 μg/mL in Ventana Avidin Diluent withB5 Blocker (VMSI #90040). The MsAntiV5 antibody was incubated at 37° C.for 8 minutes. The slides were rinsed 3 times with reaction bufferbefore the addition of 100 μL of ultra View HRP universal multimer (acomponent of the VMSI ultra View DAB Detection Kit #760-500). After 3rinses with reaction buffer, 100 μL of both the ultra View DAB and ultraView H20 2 were applied to the slide and co-incubated for 8 minutes withLCS at 37° C. The slides were rinsed once in reaction buffer before 100μL of the Ultra View Copper was applied to the slide and incubated for 4minutes at 37° C. The slides then underwent 2 rinses with reactionbuffer before counterstaining with Hematoxylin II (VMSI #750-2021) whichwas incubated on the slide for 4 in minutes with LCS. After 2 rinseswith reaction buffer, the bluing reagent (VMSI #760-2037) was appliedand incubated for 4 minutes for the counterstain to be complete. Theslides were removed from the instrument and treated to a detergent wash,rinsed with water, dehydrated with an alcohol rundown and xylene beforemanual application of a solid cover slip. Results discussed below arefor IHC staining of a 2+ HER2+ breast cancer tissue case. The slideswere viewed through a brightfield microscope. The results shown in Table11 were a subjective score of the DAB IHC signal strength (e.g., theintensity of the staining) with 2 being the most the DAB intensityobserved for the HER2/neu (4B5) standard slide.

TABLE 11 Subjective score of the DAB IHC signal strength Antibody IHCIntensity 4B5 Control 2 H0K4 2 H0K3 1.5 H0K2 1.5 H0K1 1 H5K0 2 H4K0 2H3K0 2.25 H2K0 2 H1K0 1.75 H4K4 2.25 H3K3 2.5 H2K2 2.25

The HOK4 HER2-V5 conjugate provided comparable IHC DAB staining to theHER2/neu (4B5) standard control on the 2+ breast cancer case. The HOK1to HOK3 HER2-V 5 conjugates demonstrated slightly inferior IHC staining.The H2KO to H5KO HER2-V5 conjugates provided comparable to the HER2/neu(4B5) standard control on the 2+ breast cancer case. The H1KO HER2-V5conjugate also demonstrated slightly inferior IHC tissue staining.

Example 9: Evaluation of HER2/neu (4B5) Antibody—V5 Peptide ConjugatesHER2+ 4111 Cell Line Models and Breast Cancer Tissue

This example demonstrates the visualization of HER2/neu (4B5)antibody-V5 peptide conjugates on HER2+ 4nl cell line models and HER2+breast cancer tissue. Slides containing tonsil tissue sections weredeveloped using a standard protocol for an automated stainer [BenchMark®XT, Ventana Medical Systems, Inc. (VMSI) Tucson, Ariz.]. A typicalautomated protocol is as follows: The paraffin-coated tissue on theslides was heated to 75° C. for 4 minutes and treated once with EZPrep(VMSI #950-102), volume adjusted at 75° C. before application of theLiquid Cover Slip (LCS, VMSI #650-010). After the slide was incubatedfor 4 minutes at 75° C., the slide was rinsed and EZPrep volume wasadjusted, followed with LCS to deparaffinize the tissue (processrepeated three times—4 total cycles). The slides were cooled to 37° C.and incubated for 4 minutes. The slides were thoroughly rinsed with cellcondition solution (CCI, VMSI #950-124), followed by application of LCS.The slides were heated to 95° C. for 8 minutes. The slides were thenheated to 100° C. and incubated for 8 minutes. The slides were rinsedwith CCI followed by application of LCS and incubated for 8 minutes at100° C. Every 4 minutes, for 16 minutes, CCI and LCS were applied inorder to prevent slide drying.

The slides were cooled to 37° C. and rinsed twice with reaction buffer(VMSI #950-300), 100 μL of UV Inhibitor (a component of the VMSI ultraView DAB Detection Kit #760-500) was applied to the slide and incubatedfor 4 minutes. The slides were rinsed once with reaction buffer beforethe application of 100 μL of HER2/neu antibody-V5 peptide conjugate (1μg/mL) in Ventana Antibody Diluent with Casein (VMSI #760-219) for 16minutes at 37° C. The slides were rinsed 3 times with reaction bufferbefore the addition of 100 μL of Pierce MsAntiV5 Antibody (CloneE10N4RR, Pierce #MA5-15253) at 1 μg/mL in Ventana Avidin Diluent with B5Blocker (VMSI #90040). The MsAntiV5 antibody was incubated at 37° C. for8 minutes. The slides were rinsed 3 times with reaction buffet beforethe addition of 100 μL of ultra View HRP universal multimer (a componentof the VMSI ultra View DAB Detection Kit #760-500). After 3 rinses withreaction buffer, 100 μL of both the ultra View DAB and ultra View H20 2were applied to the slide and co-incubated for 8 minutes with LCS at 37°C. The slides were rinsed once in reaction buffer before 100 μL of theUltra View Copper was applied to the slide and incubated for 4 minutesat 37° C. The slides then underwent 2 rinses with reaction buffer beforecounterstaining with Hematoxylin II (VMSI #750-2021) which was incubatedon the slide for 4 minutes with LCS. After 2 rinses with reactionbuffer, the bluing reagent (VMSI #760-2037) was applied and incubatedfor 4 minutes for the counterstain to be complete. The slides wereremoved from the instrument and treated to a detergent wash, rinsed withwater, dehydrated with an alcohol rundown and xylene before manualapplication of a solid cover slip.

Results discussed below are for IHC staining of a 2+ HER2+ breast cancertissue case. The slides were viewed through a brightfield microscope.The results shown in Table 12 were a subjective score of the DAB IHCsignal strength (e.g., the intensity of the staining) with 2 being themost the DAB intensity observed for the HER2/neu (485) standard slide.

TABLE 2 Subjective score of the DAB IHC signal strength Antibody IHCIntensity 4B5 Control 2 H0K4 2 H0K3 1.5 H0K2 1.5 H0K1 1 H5K0 2 H4K0 2H3K0 2.25 H2K0 2 H1K0 1.75 H4K4 2.25 H3K3 2.5 H2K2 2.25

The HOK4 HER2-V5 conjugate provided comparable IHC DAB staining to theHER2/neu (4B5) standard control on the 2+ breast cancer case. The HOK1to HOK3 HER2-V5 conjugates demonstrated slightly interior IHC staining.The H2KO to H5KO HER2-V 5 conjugates provided comparable to the HER2/neu(4B5) standard control on the 2+ breast cancel case. The H1KO HER2-V5conjugate also demonstrated slightly inferior IHC tissue staining.

Example 10: BioLayer Interferometry (BL]) Evaluation of HER2/neu (4B5)Antibody—VS Peptide Conjugates: Conjugate Recognition of HER2 PeptideAntigen and Subsequent Conjugate Recognition by AntiVS Antibody

All BioLayer Interferometry experiments were performed on a ForteBioOctet Red BU instrument using ForteBio Amine Reactive Sensors (ForteBio#18-5001) in Greiner Bio-one black flat well 96-well polypropylenemicroplates (Greiner Bio-one #655209). All BLI assays were performed at37° C.

BLI analyses were performed as followed:

ForteBio Amine-reactive biosensors (ForteBio) were hydrated in 200 μL of0.1 MMES pH 5.0 [2-(N-morpholino ethanesulfonic acid)]. Biosensors wereactivated for 5 minutes by treatment in a 200 μL mixture of 0.4 M EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), 0.1 M NHS(N-hydrosulfosuccinimide) in ddi water. BSA-HER2 peptide immunogenloading was performed by treating the sensors for 20 minutes with 200 μLof BSA-HER2 peptide immunogen (25 μg/mL) in O.IM MES pH 5.0. The sensorswere quenched with 1M ethanolamine (pH=8.5) for 5 minutes followed byequilibrium in 10 mM phosphate (pH=7.4), 134 mM NaCl containing ForteBioKinetics Buffer Additive (ForteBio #18-5032 diluted to IX) for 5minutes. HER2-V5 antibody peptide conjugate binding was performed bytreating sensors for approximately 45 minutes with a 3 μg/mL solution ofthe conjugate in 10 mM phosphate (pH=7.4), 134 mM NaCl containingForteBio Kinetics Buffer Additive (ForteBio #18-5032 diluted to IX). TheHER2-V5 antibody-peptide antibody conjugate was allowed to dissociatefrom the sensor for 20 minutes in in 10 mM phosphate (pH=7.4), 134 mMNaCl containing ForteBio Kinetics Buffer Additive (ForteBio #18-5032diluted to IX). MsAntiV5 antibody recognition of the biosensor wasperformed by treating sensors for approximately 45 minutes with a 3μg/mL solution of Pierce MsAntiV5 Antibody (Clone E10/V4RR, Pierce #MAS-15253) in 10 mM phosphate (pH 7.4), 134 mM NaCl containing ForteBioKinetics Buffer Additive (ForteBio #18-5032 diluted to IX). The HER2-V5antibody-peptide antibody conjugate was allowed to dissociate from thesensor for approximately 45 minutes in 10 mM phosphate (pH=7.4), 134 mMNaCl containing ForteBio Kinetics Buffer Additive (ForteBio #18-5032diluted to IX).

Results

BioLayer Interferometry experiments were performed to demonstrate thepeptide labeling impact on antibody-peptide conjugate/antigenrecognition and then subsequently secondary anti-peptideantibody/antibody-peptide conjugate recognition. BLI studiesdemonstrated that peptide labeling of the antibody heavy and lightchains minimally impacted antibody recognition of the requisite antigen.Further studies showed that increased peptide labeling changed the modeof secondary antibody recognition from affinity to avidity interactions.Increased labeling of both heavy and light chains caused at least someassay layer compression and increased avidity, whereas secondaryantibody dissociation was greatly minimized (see FIGS. 24A and 24B).

BLI analysis showed (1) good signal/noise for all antibody recognitionsteps; (2) consistent assay response with replicate data points; and (3)no cross-reactivity was observed for MsAntiV5 with native HER2(4B5).

Example 11: DLS/DSC Testing Methods

DLS: hydrodynamic radius, aggregates, temperature induced aggregation

DSC: influence of E4 tag on melting temperature IgG domains (see Table13).

TABLE 13 Sample Conc [mg/ml] Amount [mg] CD3(SP162), E, H4K4 0.61 1.8CD3(SP162), E, HOK4 1.34 4.0 CD3(SP162), E, H4KO 0.91 1.8 CD3(SP162),native 0.96 1.0

All samples were in PBS with 0.1% sodium azide and 1% BSA; the E4 tagwas fused at C-terminus of H and/or K chain (see FIG. 25A). Themolecular weight of the E4 tag was 20 kDa.

BSA removal with Protein A chromatography:

(1) Protein A chromatography—Column: HiTrap MabSelect SuRe (1 ml);Buffer A: 50 mM KP, 150 mM NaCl pH 7.5; Buffer B: 50 mM Natriumcitrat pH4.0

(2) Dialysis for 16 h at 4° C. against 50 mM KP-Puffer 150 mM KCl pH 7.4

(3) concentrate to 0.5 mg protein/ml with Pall Macrosep Advance 10 kDa

Yield (see Table 14):

TABLE 14 Sample H4K4 H0K4 H4K0 native Start amount (specified 1.8 4.01.8 1.0 by Ventana) [mg] Amount after 0.7 1.5 0.5 0.3 concentration [mg]% yield 39 38 29 32

FIG. 25B shows that BSA was quantitatively removed from the samples (SDSpage).

SEC Chromatography—Molecular weight calculated from refractive index andright angle light scattering detector in combination with SEC (YMC 200)(see Table 15):

TABLE 15 Sample Mw-(kDa) H0K0 154 H0K4 205 H4K0 205 H4K4 252

SEC chromatography indicated that molecular weights were as expected.

DLS Measurements—Analysts and Interpretation (see Tables 16A and 16B):

TABLE 16A Hydrodynamic Radius Peak 1 (Regularization Fit) rH [nm] SampleN mean SD BSA 10 mg/ml 3 4.0 0.1 BSA 1 mg/ml 3 3.8 0.1 V184 H0K0 3 5.40.4 V184 H0K4 3 7.8 0.2 V184 H4K0 3 8.3 0.5 V184 H4K4 3 9.3 0.3

TABLE 16B Polydispersity of Peak 1 Pd [%] Sample N mean SD BSA 10 mg/ml3 15.2 3.3 BSA 1 mg/ml 3 10.7 2.1 V184 H0K0 3 12.5 3.6 V184 H0K4 3 15.52.3 V184 H4K0 3 21.6 7.5 V184 H4K4 3 12.7 1.1

Reproducibility of the measurements is good;

H0K0 had a rH (5.4 nm) as expected for an IgG, only little amount ofaggregates were detectable;

C-terminal fusion of E4 tag caused a significant increase in rH, thatcould be explained by the unstructured nature of the E4 tag;

Slightly more aggregates were detectable in the samples with E4 tag; and

H4K0 had significantly more aggregates (approx. 2% (w/w)) than theothers and a higher polydispersity in Peak 1, indicating a highercontent on oligomers (dimer to tetramer).

DLS Temperature ramp (see FIGS. 25D and 25E)

Hydrodynamic Radius (Cumulative fit) is followed over the temperaturerange from 25° C. to 80° C. with a ramp rate of 0.1 K/min (approx. 10h); the native IgG (H0K0) forms very large (˜1 μm) aggregates at 70° C.;and the temperature curves suggest, that the E4 tag reduces theformation of this temperature induced aggregates.

With OnsetTemp the starting point of aggregation was determined.OnsetTemp is a measure for the stability of the protein structure;Fusion of E4 tag to the C-terminus of the H chain has no significantinfluence on OnsetTemp; Fusion of E4 tag to the C-terminus of the Kchain causes a decrease of OnsetTemp by 2 K; this effect could beobserved at H0K4 and H4K4. The K4 fusion had a destabilizing effect onthe IgG.

DSC analysis (see FIG. 25F)—Due to the limited amount of sample, the DSCexperiments had to performed at very low concentrations as singlemeasurements; fusion of E4 tag to the C-terminus of the K chain caused asignificant decrease of Tm compared to H4K0; the destabilizing effect ofthe K4 fusion on IgG shown in the DLS experiment could be verified byDSC.

DLS/DSC Summary

All experiments were performed in 50 mM potassium phosphate buffer, 150mM potassium chloride pH 7.4. Evaluation of the effect of buffer, pH,salt concentration was not the aim of this study.

Observed effects of the E4 tag.

Increase of molecular weight was determined by RALS from 154 kDa (H0K0)to 205 kDa (H4K0 and H0K4) and 252 kDa (H4K4)

Increase of r_(H) from 5.4 nm (H0K0) to 8 nm (H4K0 and H0K4) and 9 nm(H4K4)

E4 tag at the K chain but not at the H chain reduced the stability ofthe IgG as shown by DLS OnsetTemp determination and by DSC

E4 tag reduced the tendency to form large aggregates at hightemperature.

Example 12—Kinetics and Association/Dissociate Rate Studies Overview ofSurface Plasmon Resonance Spectroscopy

Surface plasmon resonance (SPR) spectroscopy is a technique for thestudy of ligand binding interactions. SPR is capable of measuringreal-time quantitative binding affinities and kinetics for antibodiesinteracting with ligand molecules using relatively small quantities ofmaterials. The conventional SPR technique requires one binding componentto be immobilized on a sensor chip whilst the other binding component insolution is flowed over the sensor surface; a binding interaction isdetected using an optical method that measures small changes inrefractive index at the sensor surface. This exploits the phenomenon ofsurface plasmon generation in thin metal films and the total internalreflection of light at a surface-position interface to produce anelectromagnetic film or evanescent wave that extends a short distance(up to 300 nm) into the solution.

FIG. 30 denotes a schematic illustration of the basic SPR experiment formeasuring the binding of an analyte molecule to a receptor molecule. Byway of example: A. Instrument set up for an SPR experiment based onBIAcore™ technology. SPR uses an optical method to measure therefractive index near to a sensor surface; this exploits total internalreflection of light at a surface-solution interface to produce anelectromagnetic field or evanescent wave that extends a short distance(up to 300 nm) into the solution. The surface is a thin film of gold ona glass support that forms the floor of a small-volume (less than 100nl) flow cell through which an aqueous solution is continuously passed.In order to detect the binding of an analyte molecule to a receptormolecule, the receptor molecule is usually immobilized on the sensorsurface and the analyte molecule is injected in the aqueous solutionthrough the flow cell. Polarized light from a laser source is directedthrough a prism to the under surface of the gold film where surfaceplasmons are generated at a critical angle of the incident light. Thisabsorption of light is seen as a decrease in intensity of the reflectedlight. The critical angle is dependent on the refractive index of themedium within 300 nm of the gold surface and changes when molecules bindto the surface, e.g. when analyte molecules bind to immobilized receptormolecules B. Change in the critical angle of incident light from angle ato angle b on binding of an analyte molecule to a receptor molecule. C.Response of the SPR experiment in the form of a sensorgram. Ifinteraction between the immobilized receptor molecule and the analytemolecule occurs, the refractive index at the surface of the gold filmchanges and this is seen as an increase in signal intensity. Resonanceor response units (RU) are used to describe the increase in the signal,where 1 RU is equal to a critical angle shift of 10-4 deg. At the startof the experiment all immobilized receptor molecules have not beenexposed to analyte molecules and the RU correspond to the startingcritical angle a. Analyte molecules are injected into the flow cell; ifthey bind to the immobilized receptor molecules, there is an associationphase during which binding sites become occupied and the shape of thiscurve can be used to measure the rate of association (kon). When asteady-state is achieved (all binding sites occupied in this example)the RU correspond to the changed final critical angle b. This maximum RUrelates to the concentrations of immobilized receptor and analytemolecules and so can be used to measure the binding affinity (KD). Whenanalyte molecules are removed from the continuous flow there is adissociation phase during which binding sites become unoccupied and theshape of this curve can be used to measure the rate of dissociation(koff). The surface can then be regenerated and returned to the criticalangle a to start the experiment again.

Kinetics Studies

A Biacore T200 instrument (GE Healthcare) was used to kinetically assessthe binding behavior of rabbit monoclonal antibodies towards singlychemically biotinylated peptidic 2 kDa analytes:E_Tag(1-13)[Glu(Bi-PEG)-1]amid, and E_Tag(1-13)[Glu(Bi-PEG)-13]amid;E2_Tag(1-14)[Glu(Bi-PEG)-1]amid, and E2_Tag(1-12)[Glu(Bi-PEG)-12]amid;V5_Tag(1-14)[Glu(Bi-PEG)-1]amid, and V5_Tag(1-14)[Glu(Bi-PEG)-14]amid;VSV-G_Tag(1-11)[Glu(Bi-PEG)-11]amid; HA_Tag(1-9)[Glu(Bi-PEG)-1]amid, andHA_Tag(1-9)[Glu(Bi-PEG)-9]amid.

The rabbit IgG (150 kDa) monoclonal antibodies mAb<E>rRb-J26_wt-IgG andmAb<E>rRb-J26_H2L5, mAb<E2>rRb-J78_wt-IgG and mAb<E2>rRb-J78_H5L3,mAb<V5>rRb-J53_wt-IgG and mAb<V5>rRb-J53_H1L2, mAb<HA>rRb-J15_H2L2,mAb<VSV-G>rRb-J110_H5L2, and Rb-N-IgG (rabbit normal IgG, Sigma) wereinvestigated for their binding kinetics and binding stoichiometries. ACM5 series sensor was mounted into the system and was normalized inHBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/vTween 20) according to the manufacturer's instructions. The samplebuffer was the system buffer supplemented with 1 mg/ml CMD(Carboxymethyldextran, Sigma #86524). The system operated at 37° C.13000 RU GARbFcγ (goat anti rabbit Fcγ). Code Nr.: 111-005-046, JacksonImmuno Research were immobilized according to the manufacturer'sinstructions using EDC/NHS chemistry on all four flow cells. The sensorwas deactivated using 1 M ethanolamine. The binding activities of therespective antibodies against the analytes were kinetically tested.Antibodies were captured at 30 nM concentration by a 2 min injection at10 μl/min. The flow rate was set to 60 μl/min. Analytes were injectedfor 3 min at different concentration steps of 0 nM buffer control, 22nM, 67 nM, 200 nM twice, 600 nM and 1800 nM. The dissociation wasmonitored for 5 min. After each analyte injection the antibody capturesystem was fully regenerated by a 1 min 25 sec injection of HBS-ETbuffet at 20 μl/min, followed by a 1 min injection at 20 μg/min with 10nM glycine buffer pH 1.5 and two injections for 1 min at 20 μl/min with10 mM glycin pH 1.7. Where possible, kinetic signatures were evaluatedaccording to a Langmuir fit with RMAX local. In cases, where thedissociation rate was not apparent a steady state algorithm was appliedaccording to the manufacturer's evaluation software.

FIG. 31A illustrates the results of a SPR experiment in accordance withthe method described above. In particular, FIG. 31A illustrates thedifference in kinetics between Mab<V5>rRb-J53_wt andMab<V5>rRb-J53_H1L2, where MR was about 1.4, therefore indicating abouta 1:1 binding stoichiometry. There were no observable kinetic differencebetween peptides analytes labeled with biotin at position bi-1 or atposition bi-14, J53 H1L2 (one V5 tag on a heavy chain and two V5 tags onthe light chain) showed slightly higher affinity than J53 wt.

FIG. 31B illustrates the results of a SPR experiment in accordance withthe method described above. In particular FIG. 31B illustrates thedifference in kinetics between Mab<E>rRb-J26_wt and Mab<E>rRb-J26_H2L5,where MR was determined to be up to about 2, which means an approximate1:2 binding stoichiometry. Here, no determinable kd complex stabilitywas observed, but steady state affinity was noted. The E epitope tagwith biotin at position bi-1 possessed regular fast on/fast offkinetics, while the E epitope tag with biotin at position bi-13 hadunspecific binding in the dissociation phase, and this was believed tobe due to weak complex stability the antibodies.

FIG. 31C illustrates the results of a SPR experiment in accordance withthe method described above. In particular, FIG. 31C illustrates thedifference in kinetics between Mab<E2>rRb-J78_wt and Mab<E2>rRb-J78_H5L,where MR was about 1.4, therefore indicating about a 1:1 bindingstoichiometry. There were no observable kinetic differences between thepeptides with biotin at position bi-1 or at position bi-12.

FIG. 31D illustrates the results of a SPR experiment in accordance withthe method described above. In particular, FIG. 31D illustrates thedifference in kinetics between Mab<HA>rRb-J15_H2L2, where MR was about1.4, therefore indicating about a 1:1 binding stoichiometry. There wereno observable kinetic differences between peptide analytes with biotinat position bi-1 or at position bi-9.

FIG. 31E illustrates the results of a SPR experiment in accordance withthe method described above. In particular, FIG. 31E illustrates thedifference in kinetics between Mab<VSV-G>rRb-J110_H5L2, where MR was upto about 2, therefore indicating about a 1:2 binding stoichiometry.

Association/Dissociation Rate Study

A Biacore T200 instrument (GE Healthcare) was used to kinetically assessthe binding behavior of rabbit monoclonal antibodies towards therespective tagged antibody analytes: CD68 HLvsv, CD68 HvsvLvsv, CD68HvsvL, CD68 HL; CD8 HL, CD8 He2L; PD-L1 SP63 He2L, PD-L1 SP63 HLe2,PD-L1 SP63 He2Le2, PD-L1 SP63 HL; _CD20 HL, CD20 HhaL, CD20 HLha, CD20HhaLha; PD-L1 SP63 HhaL, PD-L1 SP63 HLha, PD-L1 SP63 HhaLha, PD-L1 SP63HL; FoxP3 HL, FoxP3 H5v5L, FoxP3 HL5v5; FoxP3 H5v5L5v5, FoxP3 H4v5L,FoxP3 HL4v5, FoxP3 H4v.5L4v5.

The rabbit IgG (150 kDa) monoclonal antibodies mAb<E>rRb-J78_wt-IgG andmAb<E>rRb-J26_H2L5, mAb<E2>rRb-J78_wt-IgG and mAb<E2>rRb-J78_H5L3,mAb<V5>rRb-J53_wt-IgG and mAb<V5>rRb-J53_H1L2, mAb<HA>rRb-J15_H2L2,mAb<VSV-G>rRb-J110_H5L2, and Rb-N-IgG (rabbit normal IgG, Sigma) wereinvestigated for their binding kinetics and binding stoichiometries. ACM5 series sensor was mounted into the system and was normalized inHBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/vTween 20) according to the manufacturer's instructions. The samplebuffer was the system buffer supplemented with 1 mg/ml CMD(Carboxymethyldextran, Sigma #86524). The system operated at 37° C.13000 RU GARbFcγ (goat anti rabbit Fcγ), Code Nr.: 111-005-046, JacksonImmuno Research were immobilized according to the manufacturer'sinstructions using EDC/NHS chemistry on all four flow cells. The sensorwas deactivated using 1 M ethanolamine. The binding activities of therespective antibodies against the analytes were kinetically tested.Antibodies were captured at about a 30 nM concentration by a 2 mininjection at 10 μl/min. The flow rate was set to 60 μl/min. Analyteswere injected for 3 min at different concentration steps of 0 nM buffercontrol, 22 nM, 67 nM, 200 nM twice, 600 nM and 1800 nM. Thedissociation was monitored for 5 min. After each analyte injection theantibody capture system was fully regenerated by a 1 min 25 secinjection of HBS-ET buffer at 20 μl/min, followed by a 1 min injection a20 μl/min with 10 mM glycine buffer pH 1.5 and two injections for 1 minat 20 μl/min with 10 mM glycin pH 1.7. Where possible, kineticsignatures were evaluated according to a Langmuir fit with RMAX local.In cases, where the dissociation rate was not apparent a steady statealgorithm was applied according to the manufacturer's evaluationsoftware.

According to this study, Applicants have found that the epitope-taggedantibodies are all specifically bound by the anti-tag antibodies. Asnoted in FIGS. 32A through 32D, the association rates were rapid and thedissociation rates were either out of the instrument's specificationrange or were drifting positively. The kd (l/s) was therefore set toIE-05 l/s.

When compared to the kinetics study noted above (see, for example, FIGS.31A through 31E) it was apparent that the kinetics between theepitope-tagged antibodies and the anti-tag antibodies were aviditycatalyzed. The epitope-tagged antibodies displayed two epitopes andmediated affinity. Therefore, a kinetic quantification would giveavidity but not affinity. Applicants also found that the avidity factorcould be calculated by comparing the data with from the kinetic studiesset forth above. For example, as shown in FIG. 31A, Mab<V5>rRb-J53_H1L2versus bi-14 tag (kd=2.4E-03 l/s) was at least 240-fold aviditycatalyzed (2.4E-03 l/s/1E-05 l/s=240). Similarly, FIG. 31C indicatesthat Mab<E2>rRb-J78_H5L3 (kd=2.0E-02 l/s) was at least 2000-fold aviditycatalyzed (2.0E-02 l/s/IE-05 l/s=2000).

Example 13—A Flexible and Versatile Toolbox for Parallel MultiplexImmunohistochemical Detection Using Recombinant Epitope-TaggedAntibodies and Monoclonal Anti-Tag Antibodies (see FIGS. 33A Through33E) Introduction

Contextual detection of multiple biomarkers on single formalin-fixed,paraffinembedded (FFPE) slides for clinical applications remains anunmet need. Current multiplex immunohistochemical (IHC) proceduresentail successive rounds of antibody application and fluorophoreattachment followed by antibody inactivation. We have developed anautomated and largely parallel multiplex IHC approach using series ofepitope-tagged antibodies and anti-epitope antibodies conjugated tofluorophores, haptens, or enzymes, and demonstrated feasibility by5-plex fluorescent and duplex brightfield assays of markers relevant forimmuno-oncology.

Methods

DNA sequences corresponding to peptide epitope tags were fused in-frameto rabbit monoclonal antibody cDNAs for production of anti-CD3, CD8,CD68, FoxP3, and PDL1 antibodies in mammalian cells. Recombinant taggingbypasses the potential antibody inactivation associated withchemical-based tagging. Epitope-tagged primary antibodies producedidentical diaminobenzidine (DAB) staining intensity and pattern asuntagged native antibodies (data not shown). Conjugation of fluorophoresor haptens to anti-tag or anti-hapten antibodies was performed using NHSester precursors. Horseradish peroxidase (HRP) and alkaline phosphatase(AP) were conjugated to reduced antibodies via NHS-maleimide linkers.Affinity of antiepitope antibodies was assessed using biolayerinterferometry. Automated IHC of FFPE tissue sections including tumorsamples from non-small cell lung cancer (NSCLC) patients was performedon VENTANA BenchMark ULTRA platform.

Results

Multiple clones of rabbit monoclonal antibodies against each of the fiveepitope tags were conjugated and screened for retention of affinity,stability, and appropriate staining intensity and pattern. At least oneclone of each anti-epitope antibody met the functional requirements andthese were used to stain FFPE lung sections in conjunction withcocktailed epitope-tagged antibodies. Epitope-tagged antibodies weredetected using one of three detection configurations in order ofsensitivity: 1) fluor-conjugated anti-epitope antibodies, 2)hapten-conjugated anti-tag antibodies and fluor-conjugated anti-haptenantibodies, and 3) attachment of tyramide- or quinone methide-fluors totissue specimens with HRP- or AP-conjugated antiepitope antibodies (FIG.33A). Titration of antibodies and assay optimization enabled pairings ofparticular biomarkers with detection configurations to generate specificfluorescence patterns and relative intensities comparable to thoseproduced by DAB stains using untagged antibodies and HRP-conjugatedanti-species antibodies (FIGS. 33B through 33E). Two-color brightfieldstains were generated using enzyme-conjugated anti-tag antibodies andHRP- and AP-activated chromogens (data not shown).

Conclusion

We have demonstrated feasibility of automated parallel multiplex IHCusing a series of epitope-tagged antibodies and fluor-, hapten-, orenzyme-conjugated anti-tag antibodies. The approach has followingadvantages: Streamlining workflow—applying tagged antibodies andanti-tag antibody probes as cocktails significantly shortens assay time.Flexibility and versatility—a library of haptens, epitope tags,fluorophores, and enzymes can be matched to properly detect low and highabundance markets (e.g., pairing low abundance markers with the moresensitive enzyme-mediated fluor deposition) and accommodate increasingnumbers of biomarkers beyond 5-plex. Epitope preservation—unlikeapproaches based on serially applied antibodies, using tagged andanti-tag antibodies avoids the damaging effects of multiple cycles ofantibody inactivation on tissue integrity.

Example 14—Biochemical Analysis of Binding Stoichiometry Between PeptideEpitope-Tagged Antibody and Anti-Epitope Tag Antibody

This study was initiated to determine the stoichiometry of anti-tagsecondary antibodies binding to tagged primary antibodies. Thisinformation was useful in understanding the sensitivity and degree ofsignal amplification afforded by the use of fluorophore- orenzyme-conjugated anti-tag antibodies for detection of tagged primaryantibodies. Anti-tag antibodies were incubated with antibodies tagged atC-termini of immunoglobulin heavy chains (IgH) with 4 to 5 tandempeptide epitope tags separated by hydrophilic spacer segments atincreasing molar ratios from 0.5:1 to 8:1. The bound complexes wereseparated from unbound antibodies using size exclusion chromatography.For primary antibodies with 4 tandem peptide epitope tags per IgH, nounbound anti-tag antibodies were observed until a molar ratio of 6anti-tag antibodies to 1 tagged antibody was reached. For the anti-FoxP3antibody with 5 V5 epitope tags per IgH, no unbound anti-V5 antibodieswere observed until a molar ratio of 8 anti-tag antibodies to 1 taggedantibody was reached. The data indicated that at least 4 anti-tagimmunoglobulins (IgGs) could stably associate with each IgG with 4peptide epitope tags per IgH (8 tags per entire IgG). Coupled with thesurface plasmon resonance (SPR) results, which showed that anti-tagantibodies have significantly higher affinity for tagged antibodies thanfree and non-tandem tag peptides, the current study provided compellingevidence that the specific tandem arrangement of peptide epitope tagsand spacers found in the tagged primary antibodies disclosed hereincould adopt a spatial conformation that mediates strong bivalent bindingof multiple anti-tag antibodies with no steric hindrance.

To understand the biochemical basis driving the sensitivity of thetechnology as well as its limitations, it was important to characterizehow tagged antibodies interacted with anti-tag antibodies. Previous SPRexperiments have shown tighter binding between anti-tag and taggedantibodies than between anti-tag antibodies and free peptides,suggesting avidity-mediated binding between anti-tag and taggedantibodies. However, these studies did not reveal the number of anti-tagantibodies that could become bound to each tagged antibody, and thusfell short of providing a definitive answer regarding valency ofanti-tag antibody binding to tagged antibody. As shown in FIGS. 32A-32D,the anti-tag antibodies bound tagged antibodies with rapid associationkinetics and dissociation was non-existent or too slow for an instrumentto measure reliably. Such tight binding enabled size exclusionchromatography (SEC) to be used for separation of stable tagged andanti-tag antibody complexes from free tagged antibodies or free anti-tagantibodies. The objective of this study was to determine how many foldexcess anti-tag antibodies could engage tagged antibodies in stablecomplexes until no additional anti-tag antibodies could bind and eluteas free antibodies in SEC. It was believed that the stoichiometry ofbinding between anti-tag and tagged antibodies could address the valencyquestion more definitively. In addition, this information offeredinsight into the degree of signal amplification that anti-tag antibodiescould provide as detection reagents.

Materials

The following peptide epitope-tagged antibodies dissolved in PBS wereproduced and purified by Protein A resin at Spring Bioscience (see Table17);

TABLE 17 Tagged Antibody Peptide Tag Tag Configuration* xCD8(SP239) E2H4K0 xFoxP3(SP97) V5 H5K0 xFoxP3(SP97) V5 H4K0 xPDL1(SP263) E2 H4K0xCD3(SP162) E H4K0 xCD8(SP239) AU5 H4K0 xCD68(SP251) VSV-G H4K0 *Lettersin tag configuration referred to Ig heavy (H) or kappa light (K) chainwhile numbers following the letters referred to number of tandem tagrepeats fused to the C-terminus of heavy or light chains. For example,H4K0 referred to 4 tandem tag repeats on the heavy chain with no tags onthe light chain.

The following anti-tag antibodies dissolved in PBS were produced andpurified by Protein A resin at Spring Bioscience (see Table 18):

TABLE 18 Anti-Tag Antibody xE2 J78_H5L3 xV5 J53_H1L2 xVSV-G J110_H5L2  xE J26_H2L5 xE2 J78_H5L3 xV5 J53_H1L2 xAU5 J66_H3L1

EQUIPMENT—AKTA Explorer FPLC (asset tag XX, no calibration required);Superose 6 Increase 10/300 GL size exclusion column; Hybridization oven(asset tag XX, no calibration required).

Anti-tag antibodies were incubated with tagged antibodies in about0.75×PBS and about 0.25×Reaction Buffer (RB, P/N 950-300) for about 30min at about 37° C. prior to sample loading. Reaction mixture volume wasabout 0.1 mL and entire samples were loaded. Concentration of taggedantibodies was kept constant at about 0.5 μM while concentrations ofanti-tag antibodies varied from about 0.25 μM to about 4 μM to achievemolar anti-tag to tagged antibody ratios of about 0.5:1, about 1:1,about 2:1, about 4:1, about 6:1, and when necessary about 8:1. Ascontrols, anti-tag or tagged antibodies at about 0.5 μM in about0.75×PBS and about 0.25×RB were separately prepared, incubated for about30 min at about 3° C. and loaded. Fractionation in Superose 6 column wasperformed at ambient room temperature. Protein elution was monitoredusing UV absorbance at about 210, about 230, and about 280 nm.

STATISTICAL ANALYSES—It was believed that there was no need forstatistical analyses in the execution of this study.

Peptide epitope-tagged antibodies were incubated with anti-tagantibodies at varying molar ratios to determine stoichiometry of bindingbetween them. In general, as molar ratios of tagged antibodies toanti-tag antibodies increased from 0.5:1 to 6:1, increasingly largerantibody complexes with SEC elution volumes between 13.5 and 12 mLs wereobserved (FIGS. 34A through 34G). For all of the tag and anti-tag pairstested (V5, E2, E, VSV-G, and AU5) in which 4 tandem tags and spacersegments were fused to the C-termini of each heavy chain, no freeanti-tag, antibodies were observed at anti-tag:tagged antibody molarratios of 4:1 or less (FIGS. 34A through 34F). Free anti-tag antibodiesbecame visible only at molar ratios of 6:1 (FIGS. 34A through 34F).These results indicate that the higher stoichiometry of stable bindingwas approximately 4 anti-tag antibodies per tagged antibody containing atotal of 8 peptide tags (4 tags and spacers per heavy chain) regardlessof the amino acid sequence of the peptide epitope tags. When consideredtogether with data showing stronger interaction between anti-tag andtagged antibodies than between anti-tag antibodies and free peptide tags(see FIGS. 32A-32D), these results suggested that each anti-tag antibodycould interact with a tagged antibody in a bivalent fashion by binding 2peptide tags.

At 6:1 molar ratios of anti-VSV-G to CD68-VSV-G and anti-AU5 to CD8-AU5,significant amounts of slower-eluting peaks were observed that trailedthe peaks corresponding to the largest protein complexes were observed(FIGS. 34F and 34G). These slower-eluting peaks likely representedcomplexes that consisted of less than 4 anti-tag antibodies per taggedantibody. The fact that these smaller complexes were observed only athigh ratios of anti-tag antibodies to tagged antibodies was consistentwith competition among anti-tag antibodies for limited number of epitopetags resulting in weaker monovalent interaction between tagged andanti-tag antibodies.

For tagged antibody with 5 tandem tags and spacers, no free anti-tagantibodies were observed until molar ratios of anti-tag antibody totagged antibody reached 8:1 (FIG. 34G). In addition, unlike bindingbetween anti-tag antibodies and tagged antibodies with 4 tags per IgH(H4K0) in which the complexes consisted of one predominant peak (FIGS.34A through 34D for E, E2, and V5 tags) or several minor peaks thattrailed the major peak (FIGS. 34F and 34G for VSV-G and AU5 tags), thatbetween anti-V5 and anti-FoxP3-V5 with 5 tags per IgH (H5K0) resulted inat least two complexes that were significantly larger than the majorcomplex that eluted at 11.7 mL (FIG. 34G). It is believed that the peakat 8.5 mL could have represented more than one species of proteincomplexes as it corresponded to the void volume of the Superose 6Increase column. These results suggested that while the most stablecomplex between anti-V5 and H5K0-tagged anti-FoxP3-V5 antibodiesconsisted of 5 to 6 anti-tag antibodies per tagged antibody, even largercomplexes were able to form. It is possible the larger complexes couldonly form between V5-tagged antibody and anti-V5 antibody clone J53_H1L2because the affinity of thus particular antibody clone for its cognatetag was the highest of all anti-tag antibodies (see FIGS. 31A-31E). Thehigh affinity of J53_H1L2 for the V5 tag could mediate stable monovalentbinding or cross-linking of two V5-tagged antibodies.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet are incorporated herein by reference, intheir entirety. Aspects of the embodiments can be modified, if necessaryto employ concepts of the various patents, applications and publicationsto provide yet further embodiments.

Although the disclosure herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent disclosure. It is therefore understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present disclosure as defined by the appended claims.

The invention claimed is:
 1. A kit comprising: (a) at least oneepitope-tagged antibody, wherein the at least one epitope-taggedantibody comprises an antibody and at least one epitope tag construct,and (b) detection reagents for detecting the at least one epitope-taggedantibody, wherein the detection reagents comprise at least one anti-tagantibody, wherein the at least one anti-tag antibody is specific for anexpressed epitope tag of the at least one epitope-tagged antibody, andwherein the at least one epitope tag construct consists of the aminoacid sequence selected from the group consisting of SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, and SEQ ID NO:32.
 2. The kit of claim 1, whereinthe at least one anti-tag antibody comprises a fluorophore.
 3. The kitof claim 1, wherein the at least one epitope-tagged antibody comprisesat least one epitope-tagged antibody specific for FoxP3, at least oneepitope-tagged antibody specific for CD8, at least one epitope-taggedantibody specific for CD68, at least one epitope-tagged antibodyspecific for CD3, and at least one epitope-tagged antibody specific forCD20.
 4. The kit of claim 1, wherein the at least one epitope tagconstruct is expressed at a terminal end of a heavy chain antibody. 5.The kit of claim 1, wherein the at least one epitope-tagged antibody isspecific for the antigen human FoxP3.
 6. The kit of claim 5, wherein theat least one epitope tag construct consists of the amino acid sequenceof SEQ ID NO:22.
 7. The kit of claim 1, further comprising instructionsfor use.